U.S. patent number 7,566,702 [Application Number 11/234,075] was granted by the patent office on 2009-07-28 for immunostimulatory oligonucleotide multimers.
This patent grant is currently assigned to Idera Pharmaceuticals, Inc.. Invention is credited to Sudhir Agrawal, Ekambar Kandimalla, Dong Yu.
United States Patent |
7,566,702 |
Agrawal , et al. |
July 28, 2009 |
Immunostimulatory oligonucleotide multimers
Abstract
The invention provides an immunostimulatory nucleic acid. In
certain embodiments according to this aspect of the invention, the
sequence of the immunostimulatory oligonucleotide and/or immunomer
is at least partially self-complementary.
Inventors: |
Agrawal; Sudhir (Shrewsbury,
MA), Kandimalla; Ekambar (Southboro, MA), Yu; Dong
(Westboro, MA) |
Assignee: |
Idera Pharmaceuticals, Inc.
(Cambridge, MA)
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Family
ID: |
35658053 |
Appl.
No.: |
11/234,075 |
Filed: |
September 22, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060094681 A1 |
May 4, 2006 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10892550 |
Jul 15, 2004 |
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11153054 |
Jun 15, 2005 |
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60579985 |
Jun 15, 2004 |
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60559362 |
Aug 6, 2004 |
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Current U.S.
Class: |
514/44R; 514/45;
536/23.1 |
Current CPC
Class: |
A61K
39/39 (20130101); A61K 2039/55561 (20130101) |
Current International
Class: |
C07H
21/02 (20060101); A01N 43/04 (20060101); A61K
31/70 (20060101); C07H 21/04 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
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cited by other.
|
Primary Examiner: Li; Q. Janice
Assistant Examiner: Hill; Kevin K.
Attorney, Agent or Firm: Keown; Wayne A. Zucchero; Joseph C.
Keown & Zucchero, LLP
Parent Case Text
RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent
application Ser. No. 10/892,550, filed Jul. 15, 2004 and a
continuation of U.S. patent application Ser. No. 11/153,054, filed
Jun. 15, 2005, which claims the benefit of U.S. Provisional
Application Ser. No. 60/579,985, filed Jun. 15, 2004, and U.S.
Provisional Application Ser. No. 60/599,362, filed on Aug. 6, 2004.
The entire teachings of the above-referenced Applications are
incorporated herein by reference.
Claims
What is claimed is:
1. An immunostimulatory oligonucleotide having the structure
5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5'; wherein X is a
glycerol linker, o is phosphodiester linkage and G.sub.2 is
arabinoguanosine.
2. A composition comprising the oligonucleotide according to claim
1 and a physiologically acceptable carrier.
3. The composition according to claim 2, further comprising an
antibody, antisense oligonucleotide, protein, antigen, allergen,
chemotherapeutic agent or adjuvant.
4. A composition comprising the oligonucleotide according to claim
1, further comprising an antibody, antisense oligonucleotide,
protein, antigen, allergen, chemotherapeutic agent or adjuvant.
5. A method for generating an immune response in a mammal, the
method comprising administering to the mammal an immunostimulatory
oligonucleotide having the structure
5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5'; wherein X is a
glycerol linker, o is phosphodiester linkage and G.sub.2 is
arabinoguanosine.
6. The method according to claim 5, wherein the route of
administration is selected from the group consisting of parental,
oral, sublingual, transdermal, topical, intranasal, aerosol,
intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal
patch, eye drop and mouthwash.
7. The method according to claim 5, further comprising
administering an antibody, antisense oligonucleotide, protein,
antigen, allergen, chemotherapeutic agent or adjuvant.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to immune stimulation by oligonucleotide
analogs.
2. Summary of the Related Art
Tokunaga et al., J. Natl. Cancer Inst. 72 (1984) 955-96;. Pisetsky
et al.; Reich et al., Mol. Biol. Rep. 18 (1993) 217-221; Krieg et
al., Yi et al., Nature 374 (1995) 546-549 and Sato et al., Science
273 (1996) 352-354 teach that bacterial DNA, synthetic
oligodeoxynucleotides, and DNA vaccines containing unmethylated
CpG-dinucleotides in specific sequence contexts (CpG DNA) activate
the vertebrate immune system.
Krieg et al., Annu. Rev. Immunol. 20 (2002) 709-760; Dalpke et al.,
Biol. Chem. 383 (2002) 1491-1500 and Kandimalla et al., Curr. Opin.
Mol. Ther. 4 (2002) 122-129 teach that CpG DNAs induce innate
immune cells to produce ThI cytokines that promote cytotoxic T
lymphocyte (CTL) responses and production of immunoglobulins by B
cells. The immunostimulatory properties of CpG DNAs have allowed
their use as therapeutic agents for a broad spectrum of disease
indications including cancers, viral and bacterial infections,
inflammatory disorders and as adjuvant in immunotherapy.
In addition to chemical modifications, a number of structural
modifications influenced the activity of CpG DNAs. Kandimalla et
al., Nucleic Acids Res. 30 (2002) 4460-4469 teaches that CpG DNAs
that contained two freely accessible 5'-ends through a
3'-3'-linkage had greater activity than did conventional CpG DNAs
containing multiple copies of CpG motifs and a single 5'-end.
Kandimalla et al, Biochem. Biophys. Res. Commun. 306 (2003) 948-953
teaches that the presence of a secondary structure in CpG DNAs
significantly affected their activity depending on the position and
nature of the secondary structure, that the presence of a hairpin
structure at the 5'-end abrogated stimulatory activity, and that
the same structure at the 3'-end had an insignificant effect on
stimulatory activity but caused lower IL-6 secretion and
contributed to higher stability against nucleases.
There remains a need to "customize" the immune response through
modification of oligonucleotide analogs.
BRIEF SUMMARY OF THE INVENTION
In a first aspect the invention provides an immunostimulatory
oligonucleotide the sequence of which is at least partially
self-complementary. The immunostimulatory nucleic acid comprises an
oligonucleotide sequence containing at least one dinucleotide
selected from the group consisting of CpG, C*pG, C*pG* and CpG*,
wherein C is cytidine or 2'-deoxycytidine, G is guanosine or
2'-deoxyguanosine, C* is 2'-deoxythymidine,
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,
2'-dideoxy-5-halocytosine, 2'-dideoxy-5-nitrocytosine,
arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine,
2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, or other
pyrimidine nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'- deoxyinosine, or other
purine nucleoside analogs, and p is an intemucleoside linkage
selected from the group consisting of phosphodiester,
phosphorothioate, and phosphorodithioate.
In some embodiments, the immunostimulatory nucleic acid is from
about 2 to about 50 nucleotides in length. In certain embodiments
the immunostimulatory nucleic acid is from about 12 to about 26
nucleotides in length. In some embodiments, the oligonucleotides
each have from about 3 to about 35 nucleoside residues, in further
embodiments from about 4 to about 30 nucleoside residues, in even
further embodiments from about 4 to about 20 nucleoside residues.
In some embodiments, the oligonucleotides have from about 5 to
about 18, or from about 5 to about 14, nucleoside residues. As used
herein, the term "about" implies that the exact number is not
critical. Thus, the number of nucleoside residues in the
oligonucleotides is not critical, and oligonucleotides having one
or two fewer nucleoside residues, or from one to several additional
nucleoside residues are contemplated as equivalents of each of the
embodiments described above. In some embodiments, one or more of
the oligonucleotides have 11 nucleotides.
In a second aspect the invention provides an immunomer comprising
at least two oligonucleotides linked by a non-nucleotide linker,
wherein the sequences of the immunostimulatory oligonucleotides are
at least partially self-complementary. In certain embodiments
according to this aspect of the invention at least one of the
oligonucleotides contains at least one dinucleotide selected from
the group consisting of CpG, C*pG, C*pG* and CpG*, wherein C is
cytidine or 2'-deoxycytidine, G is guanosine or 2'-deoxyguanosine,
C* is 2'-deoxythymidine,
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,
2'-dideoxy-5-halocytosine, 2'-dideoxy-5-nitrocytosine,
arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine,
2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, or other
pyrimidine nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'-deoxyinosine, or other purine
nucleoside analogs, and p is an internucleoside linkage selected
from the group consisting of phosphodiester, phosphorothioate, and
phosphorodithioate.
In some embodiments, the immunostimulatory nucleic acid is from
about 2 to about 50 nucleotides in length. In certain embodiments
the iminunostimulatory nucleic acid is from about 12 to about 26
nucleotides in length. In some embodiments, the oligonucleotides
each have from about 3 to about 35 nucleoside residues, or from
about 4 to about 30 nucleoside residues, or from about 4 to about
20 nucleoside residues. In some embodiments, the oligonucleotides
have from about 5 to about 18, or from about 5 to about 14,
nucleoside residues. As used herein, the term "about" implies that
the exact number is not critical. Thus, the number of nucleoside
residues in the oligonucleotides is not critical, and
oligonucleotides having one or two fewer nucleoside residues, or
from one to several additional nucleoside residues are contemplated
as equivalents of each of the embodiments described above. In some
embodiments, one or more of the oligonucleotides have 11
nucleotides.
In a third aspect the invention provides pharmaceutical
compositions. These compositions comprise any one of the
compositions disclosed in the first and second aspects of the
invention either alone or in combination and a pharmaceutically
acceptable carrier.
In a fourth aspect the invention provides a method for generating
an immune response in a vertebrate. This method comprises
administering to the vertebrate any one of the compositions, alone
or in combination, disclosed in the first, second and third aspects
of the invention. The compositions disclosed herein can be
administered through any suitable route of administration
including, but not limited to, parenteral, oral, sublingual,
transdermal, topical, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye
drop and mouthwash.
In a fifth aspect the invention provides a method for
therapeutically treating a vertebrate having cancer, an autoimmune
disorder, airway inflammation, inflammatory disorders, skin
disorders, allergy, asthma or a disease caused by a pathogen. This
method comprises administering to the vertebrate any one of the
compositions, alone or in combination, disclosed in the first,
second and third aspects of the invention. The compositions
disclosed herein can be administered through any suitable route of
administration including, but not limited to, parenteral, oral,
sublingual, transdermal, topical, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye
drop, ear drop and mouthwash.
In a sixth aspect the invention provides a method for preventing
cancer, an autoimmune disorder, airway inflammation, inflammatory
disorders, skin disorders, allergy, asthma or a disease caused by a
pathogen in a vertebrate. This method comprises administering to
the vertebrate any one of the compositions, alone or in
combination, disclosed in the first, second and third aspects of
the invention. The compositions disclosed herein can be
administered through any suitable route of administration
including, but not limited to, parenteral, oral, sublingual,
transdermal, topical, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye
drop, ear drop and mouthwash.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B are representations of various embodiments of the
invention. In FIG. 1B, m and n are independently 0-1000 (SEQ ID NO
41).
FIG. 2 is a synthetic scheme for the parallel synthesis of
immunomers of the invention. DMTr=4,4'-dimethoxytrityl;
CE=cyanoethyl.
FIG. 3 depicts a group of representative small molecule linkers
suitable for linear synthesis of immumomers of the invention.
FIG. 4 is a synthetic scheme for the linear synthesis of immunomers
of the invention. DMTr=4,4'-dimethoxytrityl; CE=cyanoethyl.
FIG. 5 depicts a group of representative small molecule linkers
suitable for parallel synthesis of immunomers of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The issued patents, patent applications, and references that are
cited herein are hereby incorporated by reference to the same
extent as if each was specifically and individually indicated to be
incorporated by reference. In the event of inconsistencies between
any teaching of any reference cited herein and the present
specification, the latter shall prevail for purposes of the
invention.
The invention relates to the therapeutic use of oligonucleotides as
immunostimulatory agents for immunotherapy applications. The
invention also provides methods for generating, enhancing and
modifying the immune response caused by immunostimulatory compounds
used for immunotherapy applications such as, but not limited to,
treatment and/or prevention of cancer, autoimmune disorders,
asthma, respiratory allergies, food allergies, and bacteria,
parasitic, and viral infections in adult and pediatric human and
veterinary applications. Allergic asthma is a certain embodied
condition for treatment by the present methods and compounds. Thus,
the invention further provides compounds having optimal levels of
immunostimulatory effect for immunotherapy and methods for making
and using such compounds. In addition, immunostimulatory
oligonucleotides/immunomers of the invention are useful as
adjuvants in combination with DNA vaccines, antibodies, allergens,
chemotherapeutic agents, and antisense oligonucleotides.
In a first aspect the invention provides an immunostimulatory
oligonucleotide the sequence of which is at least partially
self-complementary. The immunostimulatory nucleic acid comprises an
nucleic acid sequence containing at least one dinucleotide selected
from the group consisting of CpG, C*pG, C*pG* and CpG*, wherein C
is cytidine or 2'-deoxycytidine, G is guanosine or
2'-deoxyguanosine, C* is 2'-deoxythymidine,
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,
2'-dideoxy-5-halocytosine, 2'-dideoxy-5-nitrocytosine,
arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine,
2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, or other
pyrimidine nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'-deoxyinosine, or other purine
nucleoside analogs, and p is an intemucleoside linkage selected
from the group consisting of phosphodiester, phosphorothioate, and
phosphorodithioate.
In some embodiments, the immunostimulatory oligonucleotide is from
about 2 to about 50 nucleotides in length. In certain embodiments
the immunostimulatory oligonucleotide is from about 12 to about 26
nucleotides in length. In some embodiments, the oligonucleotides
are from about 3 to about 35 nucleoside residues, or from about 4
to about 30 nucleoside residues, or from about 4 to about 20
nucleoside residues. In some embodiments, the oligonucleotides have
from about 5 to about 18, or from about 5 to about 14, nucleoside
residues. As used herein, the term "about" implies that the exact
number is not critical. Thus, the number of nucleoside residues in
the oligonucleotides is not critical, and oligonucleotides having
one or two fewer nucleoside residues, or from one to several
additional nucleoside residues are contemplated as equivalents of
each of the embodiments described above. In some embodiments, one
or more of the oligonucleotides have 11 nucleotides.
As would be recognized by one skilled in the art, the complementary
sequence of the oligonucleotides allows for intermolecular hydrogen
bonding thereby giving the oligonucleotides secondary structure.
Additional oligonucleotides can bind together thereby creating a
chain, or multimers, of oligonucleotides according to the
invention.
In a second aspect the invention provides an immunomer comprising
at least two oligonucleotides linked by a non-nucleotide linker,
wherein the sequences of the immunostimulatory oligonucleotides are
at least partially self-complementary. In certain embodiments
according to this aspect of the invention at least one of the
oligonucleotides contains at least one dinucleotide selected from
the group consisting of CpG, C*pG, C*pG* and CpG*, wherein C is
cytidine or 2'-deoxycytidine, G is guanosine or 2'-deoxyguanosine,
C* is 2'-deoxythymidine,
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2oxo-7-deaza-8-methyl-purine, 2
'-dideoxy-5-halocytosine, 2'-dideoxy-5-nitrocytosine,
arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine,
2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, or other
pyrimidine nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'- deoxyinosine, or other
purine nucleoside analogs, and p is an internucleoside linkage
selected from the group consisting of phosphodiester,
phosphorothioate, and phosphorodithioate.
In this aspect, immunostimulatory nucleic acid comprises a
structure as detailed in formula (I). Domain A-Domain B-Domain C
(I)
Domains may be from about 2 to about 12 nucleotides in length.
Domain A may be 5'-3' or 3'-5' or 2'-5' DNA, RNA, RNA-DNA, DNA-RNA
having a palindromic or self-complementary domain containing or not
containing at least one dinucleotide selected from the group
consisting of CpG, C*pG, C*pG* and CpG*, wherein C is cytidine or
2'-deoxycytidine, G is guanosine or 2'-deoxyguanosine, C* is
2'-deoxythymidine,
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,
2'-deoxy-5-halocytosine, 2'-deoxy-5-nitrocytosine, arabinocytidine,
2'-deoxy-2'-substitutedarabinocytidine, 2'-O-substituted
arabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, or other
pyrimidine nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'-deoxyinosine, or other purine
nucleoside analogs, and p is an internucleoside linkage selected
from the group consisting of phosphodiester, phosphorothioate, and
phosphorodithioate. In certain embodiments, the immunostimulatory
dinucleotide is not CpG.
In certain embodiments, Domain A will have more than one
dinucleotide selected from the group consisting of CpG, C*pG, C*pG*
and CpG*.
Domain B, as depicted by an "X" below, is a linkerjoining Domains A
and C that may be a 3'-`5` linkage, a 2'-5' linkage, a 3'-3'
linkage, a phosphate group, a nucleoside, or a non-nucleoside
linker that may be aliphatic, aromatic, aryl, cyclic, chiral,
achiral, a peptide, a carbohydrate, a lipid, a fatty acid, mono-
tri- or hexapolyethylene glycol, or a heterocyclic moiety.
Domain C may be 5'-3' or 3'-5', 2'-5' DNA, RNA, RNA-DNA, DNA-RNA
Poly I-Poly C having a palindromic or self-complementary sequence,
containing or not containing a dinucleotide selected from the group
consisting of CpG, C*pG, C*pG*, CpG*, wherein C is cytidine or
2'-deoxycytidine, G is guanosine or 2'-deoxyguanosine, C* is
2'-deoxythymidine,
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,
2'-deoxy-5-halocytosine, 2'-dideoxy-5-nitrocytosine,
arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine,
2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other
pyrimidine nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'-deoxyinosine, or other purine
nucleoside analogs, and p is an intemucleoside linkage selected
from the group consisting of phosphodiester, phosphorothioate, and
phosphorodithioate. In certain embodiments, the immunostimulatory
dinucleotide is not CpG. In some embodiments, Domain B is
preferably a non-nucleotidic linker connecting oligonucleotides of
Domain A and Domain C, which are referred to as "immunomers." In
certain embodiments, Domain C does not have the dinucleotide CpG,
C*pG, C*pG* or CpG*.
In some embodiments, the oligonucleotides contained in formula (I)
are from about 2 to about 50 nucleotides in length. In certain
embodiments the oligonucleotides contained in formula (I) are from
about 12 to about 26 nucleotides in length. In some embodiments,
the oligonucleotides each have from about 3 to about 35 nucleoside
residues, preferably from about 4 to about 30 nucleoside residues,
more preferably from about 4 to about 20 nucleoside residues. In
some embodiments, the oligonucleotides have from about 5 to about
18, or from about 5 to about 14, nucleoside residues. As used
herein, the term "about" implies that the exact number is not
critical. Thus, the number of nucleoside residues in the
oligonucleotides is not critical, and oligonucleotides having one
or two fewer nucleoside residues, or from one to several additional
nucleoside residues are contemplated as equivalents of each of the
embodiments described above. In some embodiments, one or more of
the oligonucleotides have 11 nucleotides.
A self-complementary sequence as used herein refers to a base
sequence which, upon suitable alignment, may form intramolecular
or, more typically, intermolecular basepairing between G-C, A-T,
A-U and/or G-U wobble pairs. In one embodiment the extent of
self-complementarity is at least 50 percent. For example an 8-mer
that is at least 50 percent self-complementary may have a sequence
capable of forming 4, 5, 6, 7, or 8 G-C, A-T, A-U and/or G-U wobble
basepairs. Such basepairs may but need not necessarily involve
bases located at either end of the self-complementary
immunostimulatory oligonucleotide and/or immunomer. Where nucleic
acid stabilization may be important to the immunostimulatory
oligonucleotide and/or immunomer, it may be advantageous to "clamp"
together one or both ends of a double-stranded nucleic acid, either
by basepairing or by any other suitable means. The degree of
self-complementarity may depend on the alignment between
immunostimulatory oligonucleotide and/or immunomer, and such
alignment may or may not include single- or multiple-nucleoside
overhangs. In other embodiments, the degree of self-complementarity
is at least 60 percent, at least 70 percent, at least 80 percent,
at least 90 percent, or even 100 percent.
Similar considerations apply to intermolecular basepairing between
immunostimulatory oligonucleotides and/or immunomers of different
base sequence. Thus, where a plurality of immunostimulatory
oligonucleotides and/or immunomers are used together, the plurality
of immunostimulatory oligonucleotides and/or immunomers may, but
need not, include sequences which are at least partially
complementary to one another. In one embodiment the plurality of
immunostimulatory oligonucleotides and/or immunomers includes an
immunostimulatory oligonucleotide and/or immunomer having a first
sequence and an immunostimulatory oligonucleotide and/or immunomer
having a second sequence, wherein the first sequence and the second
sequence are at least 50 percent complementary. For example, as
between two 8-mers that are at least 50 percent complementary, they
may form 4, 5, 6, 7, or 8 G-C, A-T, A-U, and/or G-U wobble
basepairs. Such basepairs may but need not necessarily involve
bases located at either end of the complementary immunostimulatory
oligonucleotides and/or immunomers. The degree of complementarity
may depend on the alignment between immunostimulatory
oligonucleotides and/or immunomers, and such alignment may or may
not include single- or multiple-nucleoside overhangs. In other
embodiments, the degree of complementarity is at least 60 percent,
at least 70 percent, at least 80 percent, at least 90 percent, or
even 100 percent.
By way of non-limiting example, in certain embodiments of this
aspect the immunostimulatory nucleic acid will have a structure as
detailed in formula (II).
##STR00001## As would be recognized by one skilled in the art, the
depicted immunostimulatory nucleic acid/immunomer compounds have
secondary structure because the sequences of the domains are
complementary allowing for intermolecular hydrogen bonding. Domains
A and A' may or may not be identical, domains A and C may or may
not be identical, domains A and C' may or may not be identical,
domains A' and C may or may not be identical, domains A' and C' may
or may not be identical, domains B and B' may or may not be
identical and domains C and C' may or may not be identical.
Moreover, as shown in FIG. 1, additional immunomers can bind
through intermolecular hydrogen bonding thereby creating a chain,
or multimers, of immunomers according to the invention. n can be
any number of continuous, self complementary immunomer
compounds.
As used herein, the term "complementary" means having the ability
to hybridize to a nucleic acid. Such hybridization is ordinarily
the result of hydrogen bonding between complementary strands,
preferably to form Watson-Crick or Hoogsteen base pairs, although
other modes of hydrogen bonding, as well as base stacking can also
lead to hybridization.
As used herein, the term "secondary structure" refers to
intermolecular hydrogen bonding. Intermolecular hydrogen bonding
results in the formation of a duplexed nucleic acid molecule.
"Palindromic sequence" shall mean an inverted repeat (i.e., a
sequence such as ABCDEE'D'C'B'A' in which A and A', B and B', etc.,
are bases capable of forming the usual Watson-Crick base pairs: In
vivo, such sequences may form double-stranded structures. In one
embodiment the CpG nucleic acid contains a palindromic sequence. A
palindromic sequence used in this context refers to a palindrome in
which the CpG is part of the palindrome. In some embodiments the
CpG is the center of the palindrome. In another embodiment the CpG
nucleic acid is free of a palindrome. An immunostimulatory nucleic
acid that is free of a palindrome is one in which the CpG
dinucleotide is not part of a palindrome. Such an oligonucleotide
may include a palindrome in which the CpG is not the center of the
palindrome.
For purposes of the invention, the term "oligonucleotide" refers to
a polynucleoside formed from a plurality of linked nucleoside
units. Such oligonucleotides can be obtained from existing nucleic
acid sources, including genomic or cDNA, but are preferably
produced by synthetic methods. In some embodiments each nucleoside
unit includes a heterocyclic base and a pentofuranosyl,
2'-deoxypentfuranosyl, trehalose, arabinose,
2'-deoxy-2'-substituted arabinose, 2'-O-substituted arabinose or
hexose sugar group. The nucleoside residues can be coupled to each
other by any of the numerous known internucleoside linkages. Such
intemucleoside linkages include, without limitation,
phosphodiester, phosphorothioate, phosphorodith ioate, al kyl
phosphonate, alkylphosphonoth ioate, phosphotriester,
phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate,
carbamate, morpholino, borano, thioether, bridged phosphoramidate,
bridged methylene phosphonate, bridged phosphorothioate, and
sulfone internucleoside linkages. The term "oligonucleotide" also
encompasses polynucleosides having one or more stereospecific
internucleoside linkage (e.g., (RP)- or (SP)-phosphorothioate,
alkylphosphonate, or phosphotriester linkages). As used herein, the
terms "oligonucleotide" and "dinucleotide" are expressly intended
to include polynucleosides and dinucleosides having any such
internucleoside linkage, whether or not the linkage comprises a
phosphate group. In certain embodiments, these internucleoside
linkages may be phosphodiester, phosphorothioate, or
phosphorodithioate linkages, or combinations thereof.
The term "oligonucleotide" also encompasses polynucleosides having
additional substituents including, without limitation, protein
groups, lipophilic groups, intercalating agents, diamines, folic
acid, cholesterol and adamantane. The term "oligonucleotide" also
encompasses any other nucleobase containing polymer, including,
without limitation, peptide nucleic acids (PNA), peptide nucleic
acids with phosphate groups (PHONA), locked nucleic acids (LNA),
morpholino-backbone oligonucleotides, and oligonucleotides having
backbone sections with alkyl linkers or amino linkers.
The oligonucleotides of the invention can include naturally
occurring nucleosides, modified nucleosides, or mixtures thereof.
As used herein, the term "modified nucleoside" is a nucleoside that
includes a modified heterocyclic base, a modified sugar moiety, or
a combination thereof. In some embodiments, the modified nucleoside
is a non-natural pyrimidine or purine nucleoside, as herein
described. In some embodiments, the modified nucleoside is a
2'-substituted ribonucleoside an arabinonucleoside or a
2'-deoxy-2'-substituted-arabinoside.
For purposes of the invention, the term "2'-substituted
ribonucleoside" or "2'-substituted arabinoside" includes
ribonucleosides or arabinonucleosides in which the hydroxyl group
at the 2' position of the pentose moiety is substituted to produce
a 2'-substituted or 2'-O-substituted ribonucleoside. In certain
embodiments, such substitution is with a lower alkyl group
containing 1-6 saturated or unsaturated carbon atoms, or with an
aryl group having 6-10 carbon atoms, wherein such alkyl, or aryl
group may be unsubstituted or may be substituted, e.g., with halo,
hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy,
carboxyl, carboalkoxy, or amino groups. Examples of
2'-O-substituted ribonucleosides or 2'-O-substituted-arabinosides
include, without limitation 2'-O-methylribonucleosides or
2'-O-methylarabinosides and 2'-O-methoxyethoxyribonucleosides or 2
-O-methoxyethoxyarabinosides.
The term "2'-substituted ribonucleoside" or "2'-substituted
arabinoside" also includes ribonucleosides or arabinonucleosides in
which the 2'-hydroxyl group is replaced with a lower alkyl group
containing 1-6 saturated or unsaturated carbon atoms, or with an
amino or halo group. Examples of such 2'-substituted
ribonucleosides or 2'-substituted arabinosides include, without
limitation, 2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl
ribonucleosides or arabinosides.
The term "oligonucleotide" includes hybrid and chimeric
oligonucleotides. A "chimeric oligonucleotide" is an
oligonucleotide having more than one type of intemucleoside
linkage. One non-limiting example of such a chimeric
oligonucleotide is a chimeric oligonucleotide comprising a
phosphorothioate, phosphodiester or phosphorodithioate region and
non-ionic linkages such as alkylphosphonate or
alkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Pat.
Nos. 5,635,377 and 5,366,878).
A "hybrid oligonucleotide" is an oligonucleotide having more than
one type of nucleoside. One non-limiting example of such a hybrid
oligonucleotide comprises a ribonucleotide or 2' substituted
ribonucleotide region, and a deoxyribonucleotide region (see, e.g.,
Metelev and Agrawal, U.S. Pat. Nos. 5,652,355, 6,346,614 and
6,143,881).
Alternatively, the nucleic acid molecule of the invention can be
two immunomers linked by way of a non-nucleotidic linker.
In certain embodiments of the invention, at least one
immunostimulatory oligonucleotide of the invention comprises an
immunostimulatory dinucleotide of the formula 5'-Pyr-Pur-3',
wherein Pyr is a natural pyrimidine nucleoside or analog thereof
and Pur is a natural purine nucleoside or analog thereof. As used
herein, the term "pyrimidine nucleoside" refers to a nucleoside
wherein the base component of the nucleoside is a pyrimidine base.
Similarly, the term "purine nucleoside" refers to a nucleoside
wherein the base component of the nucleoside is a purine base. For
purposes of the invention, a "synthetic" pyrimidine or purine
nucleoside includes a non-naturally occurring pyrimidine or purine
base, a non-naturally occurring sugar moiety, or a combination
thereof.
In certain embodiments pyrimidine nucleosides in the
immunostimulatory oligonucleotides and/or immunomers used in the
method according to the invention have the structure (III):
##STR00002## wherein:
D is a hydrogen bond donor;
D' is selected from the group consisting of hydrogen, hydrogen bond
donor, hydrogen bond acceptor, hydrophilic group, hydrophobic
group, electron withdrawing group and electron donating group;
A is a hydrogen bond acceptor or a hydrophilic group;
A' is selected from the group consisting of hydrogen bond acceptor,
hydrophilic group, hydrophobic group, electron withdrawing group
and electron donating group;
X is carbon or nitrogen; and
S' is a pentose or hexose sugar ring, or a non-naturally occurring
sugar.
In certain embodiments, the sugar ring is derivatized with a
phosphate moiety, modified phosphate moiety, or other linker moiety
suitable for linking the pyrimidine nucleoside to another
nucleoside or nucleoside analog.
In some embodiments hydrogen bond donors include, without
limitation, --NH--, --NH.sub.2, --SH and --OH. Preferred hydrogen
bond acceptors include, without limitation, C.dbd.O, C.dbd.S, and
the ring nitrogen atoms of an aromatic heterocycle, e.g., N3 of
cytosine.
In some embodiments, the base moiety in (III) is a non-naturally
occurring pyrimidine base. Examples of preferred non-naturally
occurring pyrimidine bases include, without limitation,
5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, or
N4-ethylcytosine, and 4-thiouracil. In some embodiments, the sugar
moiety S' in (III) is a non-naturally occurring sugar moiety. For
purposes of the present invention, a "naturally occurring sugar
moiety" is a sugar moiety that occurs naturally as part of nucleic
acid, e.g., ribose and 2'-deoxyribose and a "non-naturally
occurring sugar moiety" is any sugar that does not occur naturally
as part of a nucleic acid, but which can be used in the backbone
for an oligonucleotide, e.g, hexose. Arabinose and arabinose
derivatives are non-limiting examples of sugar moieties.
In some embodiments purine nucleoside analogs in immunostimulatory
oligonucleotides and/or immunomers used in the method according to
the invention have the structure (IV):
##STR00003##
wherein:
D is a hydrogen bond donor;
D' is selected from the group consisting of hydrogen, hydrogen bond
donor, and hydrophilic group;
A is a hydrogen bond acceptor or a hydrophilic group;
X is carbon or nitrogen;
each L is independently selected from the group consisting of C, O,
N and S; and
S' is a pentose or hexose sugar ring, or a non-naturally occurring
sugar.
In certain embodiments, the sugar ring is derivatized with a
phosphate moiety, modified phosphate moiety, or other linker moiety
suitable for linking the pyrimidine nucleoside to another
nucleoside or nucleoside analog.
In certain embodiments hydrogen bond donors include, without
limitation, --NH--, --NH.sub.2, --SH and --OH. Preferred hydrogen
bond acceptors include, without limitation, C.dbd.O, C.dbd.S,
--NO.sub.2 and the ring nitrogen atoms of an aromatic heterocycle,
e.g., N1 of guanine.
In some embodiments, the base moiety in (IV) is a non-naturally
occurring purine base. Examples of preferred non-naturally
occurring purine bases include, without limitation, 6-thioguanine
and 7-deazaguanine. In some embodiments, the sugar moiety S' in
(IV) is a naturally occurring sugar moiety, as described above for
structure (III).
In a third aspect the invention provides pharmaceutical
compositions. These compositions comprise any one of the
compositions disclosed in the first and second of the invention
either alone or in combination and a pharmaceutically acceptable
carrier.
As used herein, the term "physiologically acceptable" refers to a
material that does not interfere with the effectiveness of the
compositions of the first, second or third aspects of the invention
and is compatible with a biological system such as a cell, cell
culture, tissue, or organism. In certain embodiments, the
biological system is a living organism, such as a vertebrate.
As used herein, the term "carrier" encompasses any excipient,
diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or
other material well known in the art for use in pharmaceutical
formulations. It will be understood that the characteristics of the
carrier, excipient, or diluent will depend on the route of
administration for a particular application. The preparation of
pharmaceutically acceptable formulations containing these materials
is described in, e.g., Remington's Pharmaceutical Sciences, 18th
Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990,
ISBN: 0-912734-04-3.
Pharmaceutical compositions of the invention may also include a
cancer vaccine, including a cancer vaccine selected from EFG,
Anti-idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine,
MGV ganglioside conjugate vaccine, Her2/new, Ovarex, M-Vax, O-Vax,
L-Vax, STn-KHL theratope, BLP25 (MUC-1), liposomal idiotypic
vaccine, Melacine, peptide antigen vaccines, toxin/antigen
vaccines, MVA-vased vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN,
DISC-virus and ImmunCyst/TheraCys.
In various embodiments of the invention, the compositions of the
first, second or third aspects of the invention may be covalently
linked to an antigen or otherwise operatively associated with an
antigen. As used herein, the term "operatively associated with"
refers to any association that maintains the activity of both the
compositions of the first, second or third aspects of the invention
and the antigen. Non-limiting examples of such operative
associations include being part of the same liposome or other such
delivery vehicle or reagent. In embodiments wherein the
compositions of the first, second or third aspects of the invention
are covalently linked to an antigen, such covalent linkage is at
any position on the compositions of the first, second or third
aspects of the invention other than an accessible 5' end of an
immunostimulatory oligonucleotide. For example, the antigen may be
attached at an intemucleoside linkage or may be attached to the
non-nucleotidic linker. Alternatively, the antigen may itself be
the non-nucleotidic linker.
In various embodiments of the invention, the compositions of the
first, second or third aspects of the invention may include an
oligonucleotide with antisense activity. As used herein, "antisense
activity" means that the oligonucleotide, when introduced into a
cell or an animal, causes a reduction in the expression of the gene
to which it is complementary.
In various embodiments of the invention, the compositions of the
first, second or third aspects of the invention may include an
oligonucleotide sequence that is an aptamer. Aptamers are nucleic
acid molecules that have been selected from random pools based on
their ability to bind other molecules. Aptamers have been selected
which bind nucleic acids, proteins, small organic compounds, and
even entire organisms. These novel molecules have many potential
uses in medicine and technology (see, e.g., Burgstaller P., et al.
Curr Opin Drug Discov Devel. 5: 690-700 (2002)).
The pharmaceutical compositions of the invention may be
administered by any suitable route, including, without limitation,
parenteral, oral, sublingual, transdermal, topical, intranasal,
aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene
gun, dermal patch or in eye drop or mouthwash form. The
pharmaceutical compositions can be delivered using known procedures
at dosages and for periods of time effective obtain the desired
effect, e.g. the treatment of cancer, the treatment of infection
and the treatment of autoimmune diseases. When administered
systemically, the pharmaceutical compositions are administered at a
sufficient dosage to attain a blood level of the compositions of
the first, second and/or third aspects of the invention from about
0.0001 micromolar to about 10 micromolar. For localized
administration, much lower concentrations than this may be
effective, and much higher concentrations may be tolerated. In
certain embodiments, a total dosage of immunostimulatory
oligonucleotide and/or immunomer ranges from about 0.0001 mg per
patient per day to about 200 mg per kg body weight per day. It may
be desirable to administer simultaneously, or sequentially a
therapeutically effective amount of one or more of the therapeutic
compositions of the invention to an individual as a single
treatment episode.
Immunostimulatory oligonucleotides were created as immunomers using
the following protocols for synthesis. The immunostimulatory
oligonucleotides and/or immunomers of the invention may
conveniently be synthesized using an automated synthesizer and
phosphoramidite approach as schematically depicted in FIGS. 2 and
4. In some embodiments, the immunostimulatory oligonucleotides
and/or immunomers are synthesized by a linear synthesis approach
(see FIG. 2). Representative linkers for this synthesis are
presented in FIG. 3. As used herein, the term "linear synthesis"
refers to a synthesis that starts at one end of the immunomer and
progresses linearly to the other end. Linear synthesis permits
incorporation of either identical or un-identical (in terms of
length, base composition and/or chemical modifications
incorporated) monomeric units into the immunostimulatory
oligonucleotides and/or immunomers.
An alternative mode of synthesis for immunostimulatory
oligonucleotides and/or immunomers is "parallel synthesis", in
which synthesis proceeds outward from a central linker moiety (see
FIG. 4). Representative linkers for this method of synthesis are
presented in FIG. 5. A solid support attached linker can be used
for parallel synthesis, as is described in U.S. Pat. No. 5,912,332.
Alternatively, a universal solid support, such as phosphate
attached to controlled pore glass support, can be used.
Parallel synthesis of immunostimulatory oligonucleotides and/or
immunomers has several advantages over linear synthesis: (1)
parallel synthesis permits the incorporation of identical monomeric
units; (2) unlike in linear synthesis, both (or all) the monomeric
units are synthesized at the same time, thereby the number of
synthetic steps and the time required for the synthesis is the same
as that of a monomeric unit; and (3) the reduction in synthetic
steps improves purity and yield of the final immunomer product.
At the end of the synthesis by either linear synthesis or parallel
synthesis protocols, the immunostimulatory oligonucleotides or
immunomers according tothe invention may conveniently be
deprotected with concentrated ammonia solution or as recommended by
the phosphoramidite supplier, if a modified nucleoside is
incorporated. The product immunostimulatory oligonucleotides and/or
immunomer is preferably purified by reversed phase HPLC,
detritylated, desalted and dialyzed.
The compositions disclosed in the first second and third aspects of
the invention can comprise the immunostimulatory oligonucleotide or
immunomer alone or as oligonucleotide/immunomer conjugates. An
oligonucleotide/immunomer conjugate comprises an oligonucleotide or
immunomer, as described above, and an antigen conjugated to the
oligonucleotide and/or immunomer at a position other than the
accessible 5' end. In some embodiments, the non-nucleotidic linker
comprises an antigen, which is conjugated to the oligonucleotide.
In some other embodiments, the antigen is conjugated to the
oligonucleotide at a position other than its 3' end. In some
embodiments, the antigen produces a vaccine effect. The
immunostimulatory oligonucleotide or immunomer alone or as
oligonucleotide/immunomer conjugates can be administered in the
methods discussed below.
The antigen is optionally selected from antigens associated with a
pathogen, antigens associated with a cancer, antigens associated
with an auto-immune disorder, and antigens associated with other
diseases such as, but not limited to, veterinary or pediatric
diseases, or wherein the antigen is an allergen. For purposes of
the invention, the term "associated with" means that the antigen is
present when the pathogen, cancer, auto-immune disorder, food
allergy, skin allergy, respiratory allergy, asthma or other disease
is present, but either is not present, or is present in reduced
amounts, when the pathogen, cancer, auto-immune disorder, food
allergy, skin allergy, respiratory allergy, or disease is
absent.
The immunomer is covalently linked to the antigen, or it is
otherwise operatively associated with the antigen. As used herein,
the term "operatively associated with" refers to any association
that maintains the activity of both immunomer and antigen.
Nonlimiting examples of such operative associations include being
part of the same liposome or other such delivery vehicle or
reagent. In embodiments wherein the immunomer is covalently linked
to the antigen, such covalent linkage preferably is at any position
on the immunomer other than an accessible 5' end of an
immunostimulatory oligonucleotide. For example, the antigen may be
attached at an intemucleoside linkage or may be attached to the
non-nucleotidic linker. Alternatively, the antigen may itself be
the non-nucleotidic linker.
In a fourth aspect, the invention provides methods for generating
and/or modulating an immune response in a vertebrate, such methods
comprising administering to the vertebrate an immunomer or
immunomer conjugate according to the invention. In some
embodiments, the vertebrate is a mammal. For purposes of this
invention, the term "mammal" is expressly intended to include
humans. In certain embodiments, the immunomer or immunomer
conjugate is administered to a vertebrate in need of
immunostimulation.
As used herein, the term "modulating" or "modulate" means to
increase or decrease the immunostimulatory activity of an
immunostimulatory nucleic acid relative to that of the parent
immunostimulatory nucleic acid.
In the methods according to this aspect of the invention,
administration of immunomers can be by any suitable route,
including, without limitation, parenteral, oral, sublingual,
transdermal, topical, intranasal, intramuscular, intraperitonal,
subcutaneous, intradermal, aerosol, intraocular, intratracheal,
intrarectal, vaginal, by gene gun, dermal patch or in eye drop or
mouthwash form. Administration of the therapeutic compositions of
immunomers can be carried out using known procedures at dosages and
for periods of time effective to reduce symptoms or surrogate
markers of the disease. When administered systemically, the
therapeutic composition is preferably administered at a sufficient
dosage to attain a blood level of immunomer from about 0.0001
micromolar to about 10 micromolar. For localized administration,
much lower concentrations than this may be effective, and much
higher concentrations may be tolerated. Preferably, a total dosage
of immunomer ranges from about 0.001 mg per patient per day to
about 200 mg per kg body weight per day. It may be desirable to
administer simultaneously, or sequentially a therapeutically
effective amount of one or more of the therapeutic compositions of
the invention to an individual as a single treatment episode.
Either the immunomer or the vaccine, or both, may optionally be
linked to an immunogenic protein, such as keyhole limpet hemocyanin
(KLH), cholera toxin B subunit, or any other immunogenic carrier
protein or nonimmunogenic carrier protein. Any of the plethora of
adjuvants may be used including, without limitation, Freund's
complete adjuvant, Freund's incomplete adjuvant, KLH,
monophosphoryl lipid A (MPL), alum, and saponins, including QS-21,
imiquimod, R848, or combinations thereof.
Toll-like receptors (TLRs) function as sensors of infection and
induce the activation of innate and adaptive immune responses. TLRs
recognize a wide variety of ligands, called pathogen-associated
molecular patterns (PAMPs). Upon recognizing conserved
pathogen-associated molecular products, TLRs activate host defense
responses through their intracellular signaling domain, the
Toll/interleukin-1 receptor (TIR) domain, and the downstream
adaptor protein MyD88. Dendritic cells and macrophages normally
respond to Toll-like receptor (TLR) ligands and cytokines (for
example, interleukin-1.beta.; IL-6 and tumor necrosis factor, TNF),
which they also produce; natural killer (NK) cells and T cells are
also involved in the pro-inflammatory circuit. After TLR
stimulation by bacterial compounds, innate immune cells release a
range of cytokines. Some examples of TLR ligands include, but are
not limited to, lipoproteins; peptidoglycan, zymosan (TLR2),
double-stranded RNA, polyl:polyc (TLR3), lipopolysaccharide, heat
shock proteins, taxol (TLR4), flagellin (TLR5), and
imidazoquinolines- R848, resiquimod, imiquimod; ssRNA (TLR7/8),
beta-lymphocytes (TLR10) and uropathogenic E. coli (TLR11).
The methods according to this aspect of the invention are useful
for model studies of the immune system. The methods are also useful
for the prophylactic or therapeutic treatment of human or animal
disease. For example, the methods are useful for pediatric and
veterinary vaccine applications.
In a fifth aspect, the invention provides methods for
therapeutically treating a vertebrate having a disease or disorder,
such methods comprising administering to the vertebrate an
immunomer or immunomer conjugate according to the invention. In
various embodiments, the disease or disorder to be treated is
cancer, an autoimmune disorder, airway inflammation, inflammatory
disorders, allergy, asthma or a disease caused by a pathogen.
Pathogens include bacteria, parasites, fungi, viruses, viroids and
prions. Administration is carried out as described for the fourth
aspect of the invention.
For purposes of the invention, the term "allergy" includes, without
limitation, food allergies atopic dermatitis, allergic rhinitis
(also known as hay fever), allergic conjunctivitis, urticaria (also
known as hives), respiratory allergies and allergic reactions to
other substances such as latex, medications and insect stings or
problems commonly resulting from allergic rhinitis-sinusitis,
otitis media and COPD. The term "airway inflammation" includes,
without limitation, asthma. Specific examples of asthma include,
but are not limited to, allergic asthma, non-allergic asthma,
exercised-induced asthma, occupational asthma, and nocturnal
asthma.
Allergic asthma is characterized by airway obstruction associated
with allergies and triggered by substances called allergens.
Triggers of allergic asthma include, but are not limited to,
airborne pollens, molds, animal dander, house dust mites and
cockroach droppings. Non-allergic asthma is caused by viral
infections, certain medications or irritants found in the air,
which aggravate the nose and airways. Triggers of non-allergic
asthma include, but are not limited to, airborne particles (e.g.,
coal, chalk dust), air pollutants (e.g., tobacco smoke, wood
smoke), strong odors or sprays (e.g., perfumes, household cleaners,
cooking fumes, paints or varnishes), viral infections (e.g., colds,
viral pneumonia, sinusitis, nasal polyps), aspirin-sensitivity, and
gastroesophageal reflux disease (GERD). Exercise-induced asthma
(EIA) is triggered by vigorous physical activity. Symptoms of EIA
occur to varying degrees in a majority of asthma sufferers and are
likely to be triggered as a result of breathing cold, dry air while
exercising. Triggers of EIA include, but are not limited to,
breathing airborne pollens during exercise, breathing air
pollutants during exercise, exercising with viral respiratory tract
infections and exercising in cold, dry air. Occupational asthma is
directly related to inhaling irritants and other potentially
harmful substances found in the workplace. Triggers of occupational
asthma include, but are not limited to, fumes, chemicals, gases,
resins, metals, dusts, vapors and insecticides.
As used herein, the term "autoimmune disorder" refers to disorders
in which "self" proteins undergo attack by the immune system. Such
term includes autoimmune asthma.
Without wishing to be bound to any particular theory, decreased
exposure to bacteria may be partially responsible for the increased
incidence of, severity of, and mortality due to allergic diseases
such as asthma, atopic dermatitis, and rhinitis in the developed
countries. This hypothesis is supported by evidence that bacterial
infections or products can inhibit the development of allergic
disorders in experimental animal models and clinical studies.
Bacterial DNA or synthetic oligodeoxynucleotides containing
unmethylated CpG dinucleotides and/or modified CpG dinucleotides in
certain sequence contexts (CpG DNA) potently stimulate innate
immune responses and thereby acquired immunity. The immune response
to CpG DNA includes activation of innate immune cells,
proliferation of B cells, induction of Th1 cytokine secretion, and
production of immunoglobulins (Ig). The activation of immune cells
by CpG DNA occurs via Toll-like receptor 9 (TLR9), a molecular
pattern recognition receptor. CpG DNA induce strong Th1-dominant
immune responses characterized by secretion of IL-12 and IFN-{tilde
over (.gamma.)}. Immunomers (IMO) alone or as allergen conjugates
decrease production of IL-4, IL-5, and IgE and reduce eosinophilia
in mouse models of allergic asthma. IMO compounds also effectively
reverse established atopic eosinophilic airway disease by
converting a Th2 response to a Th1 response.
OVA with alum is commonly used to establish a Th2-dominant immune
response in various mouse and rat models. The Th2 immune response
includes increased IL-4, IL-5, and IL-13 production, elevated serum
levels of total and antigen-specific IgE, IgG1, and lower levels of
IgG2a. IMO compounds prevent and reverse established Th2-dominant
immune responses in mice. The co-administration of IMO compounds
with OVA/alum to mice reduces IL-4, IL-5, and IL-13 production and
induces IFN-.gamma. production in spleen-cell cultures subjected to
antigen re-stimulation. Furthermore, IMO compounds inhibit
antigen-specific and total IgE and enhance IgG2a production in
these mice.
Injection of OVA/alum and IMO compounds induces a lymphocyte
antigen-recall response (Th1-type) in mice characterized by low
levels of Th2-associated cytokines, IgE and IgG1, and high levels
of Th1-associated cytokines and IgG2a. Co-administration of IMO
compounds with other kinds of antigens, such as S. masoni egg and
hen egg lysozyme, also result in reversal of the Th2-response to a
Th1-dominant response in in vitro and in vivo studies. As described
herein, IMO compounds effectively prevent development of a Th2
immune response and allow a strong Th1 response.
While Th2 cytokines trigger an Ig isotype switch towards production
of IgE and IgG1, the Th1 cytokine IFN-.gamma. induces production of
IgG2a by B-lymphocytes. Mice injected with OVA/alum and IMO
compounds produce lower levels of IL-4, IL-5, and IL-13 and higher
levels of IFN-.gamma., accompanied by lower IgE and IgG1 and higher
IgG2a levels, than mice injected with OVA/alum alone. This suggests
the existence of a close link between Th1-cytokine induction and
immunoglobulin isotype switch in mice that receive antigen and IMO
compounds.
Serum antigen-specific and total IgE levels are significantly lower
in mice receiving OVA/alum and IMO compounds than in mice receiving
OVA/alum alone. In contrast, OVA-specific IgG1 levels are
insignificantly changed and total IgG1 levels are only slightly
decreased compared with mice injected with OVA/alum alone (data not
shown). The different response may result from different mechanisms
involved in the control of IgE and IgG1 class switch, though both
isotypes are influenced by IL-4 and IL-13. For example, IL-6
promotes B lymphocytes to synthesize IgG1 in the presence of
IL-4.
In a sixth aspect the invention provides a method for preventing
cancer, an autoimmune disorder, airway inflammation, inflammatory
disorders, skin disorders, allergy, asthma or a disease caused by a
pathogen in a vertebrate. This method comprises administering to
the vertebrate any one of the compositions, alone or in
combination, disclosed in the first, second and third aspects of
the invention. Pathogens include bacteria, parasites, fungi,
viruses, viroids and prions. Administration is carried out as
described for the fourth aspect of the invention.
In any of the methods according to the invention, the
immunostimulatory oligonucleotide and/or immunomer or a conjugate
thereof can be administered in combination with any other agent
useful for treating the disease or condition that does not diminish
the immunostimulatory effect of the oligonucleotide or immunomer.
For purposes of this aspect of the invention, the term "in
combination with" means in the course of treating the same disease
in the same patient, and includes administering the oligonucleotide
and/or immunomer and an agent in any order, including simultaneous
administration, as well as any temporally spaced order, for
example, from sequentially with one immediately following the other
to up to several days apart. Such combination treatment may also
include more than a single administration of the immunomer, and
independently the agent. The administration of the oligonucleotide
and/or immunomer and agent may be by the same or different
routes.
In any of the methods according to the invention, the agent useful
for treating the disease or condition includes, but is not limited
to, vaccines, antigens, antibodies, cytotoxic agents, allergens,
antibiotics, antisense oligonucleotides, peptides, proteins, gene
therapy vectors, DNA vaccines and/or adjuvants to enhance the
specificity or magnitude of the immune response, or co-stimulatory
molecules such as cytokines, chemokines, protein ligands,
trans-activating factors, peptides and peptides comprising modified
amino acids. Additionally, the agent can include DNA vectors
encoding for antigen or allergen. In these embodiments, the
immunomers of the invention can variously act as adjuvants and/or
produce direct immunostimulatory effects.
The examples below are intended to further illustrate certain
preferred embodiments of the invention, and are not intended to
limit the scope of the invention.
EXAMPLES
Example 1
Oligonucleotide Synthesis, Purification and Thermal Melt
Profiles
CpG oligonucleotides (immunostimulatory
oligonucleotides/immunomers) were synthesized on a 1 to 2 .mu.mole
scale using .beta.-cyanoethylphosphoramidites on a PerSeptive
Biosystem's 8909 Expedite DNA synthesizer (PerSeptive Biosystem,
Boston, Mass.). The phosphoramidites of dA, dG, dC, and T were
obtained from PE Biosystems (Foster City, Calif.). As described by
Iyer R. P., et al. (J. Am. Chem. Soc. 112: 1253-1254 (1990)), an
iodine oxidizing agent was used to obtain the phosphorothioate
backbone modification. All oligos were deprotected using standard
protocols, purified by HPLC, and dialyzed against USP quality
sterile water for irrigation. The oligos were lyophilized and
dissolved again in distilled water and the concentrations were
determined from UV absorbance at 260 nm. All oligos were
characterized by CGE and MALDI-TOF mass spectrometry (Applied
Biosystem's Voyager-DETM STR Biospectrometry.TM. Workstation) for
purity and molecular mass, respectively. The purity of full-length
oligos ranged from 90-96% with the rest being shorter by one or two
nucleotides (n-1 and n-2) as determined by CGE and/or denaturing
PAGE. All oligos contained less than <0.1 EU/mL of endotoxin as
determined by the Limulus assay (Bio-Whittaker now known as Cambrex
Bio Science Walkersville, Inc., Walkersville, Md.).
Thermal melting studies were carried out in 1 mL solution of 10 mM
disodium hydrogen phosphate, pH 7.2.+-.0.2, containing 150 mM NaCl,
and 2 mM MgCl2. The solutions were heated to 95.degree. C. for 10
min and allowed to come to room temperature slowly before being
stored at 4.degree. C. overnight. The final concentration of
oligonucleotide strand was 2.0 .mu.M. UV thermal melting
measurements were performed at 260 nm on a Perkin-Elmer Lambda 20
Spectrophotometer attached to a peltier thermal controller and a
personal computer using 1 cm path length quartz cuvettes at a
heating rate of 0.5.degree. C./min. Melting temperatures (Tm) were
taken as the temperature of half-dissociation and were obtained
from first derivative plots. Each Tm value is an average of two or
three independent experiments and the values were within
.+-.1.0.degree. C.
Example 2
Cell Culture Conditions and Reagents
Spleen cells from 4-8 week old BALB/c, C57BL/6 or C3H/HeJ mice were
cultured in RPMI complete medium as described by Zhao, Q., el al.
(Biochem Pharmacol. 51: 173-182 (1996)) and Branda, R. F., et al.
(Biochem. Pharmacol. 45: 2037-2043 (1993)). Murine J774 macrophages
(American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209) were cultured in Dulbecco's modified
Eagles medium supplemented with 10% (v/v) fetal calf serum and
antibiotics (100 IU/mL of penicillin G/streptomycin). All other
culture reagents were purchased from Mediatech (Gaithersburg,
Md.).
Example 3
Spleen Cell Proliferation Assay
Typically, mouse (Balb-C) spleen cells were cultured with immunomer
compounds at concentrations of 0.1, 1.0, and 10.0 .mu.g/ml for 48 h
and cell proliferation was determined by 3H-uridine incorporation,
as described by Zhao, Q., et al. (Biochem Pharmacol. 51: 173-182
(1996)).
Example 4
Cytokine Induction Assays
Mouse spleen or J774 cells were plated in 24-well dishes using
5.times.10.sup.6 or 1.times.10.sup.6 cells/mL, respectively. The
immunomer compounds dissolved in TE buffer (10 mM Tris-HCl, pH 7.5,
1 mM EDTA) were added to a final concentration of 0.03, 0.1, 0.3,
1.0, 3.0, or 10.0 .mu.g/mL to the cell cultures. The cells were
then incubated at 37.degree. C. for 24 hr and the supernatants were
collected for ELISA assays. The experiments were performed two or
three times for each immunomer compound and in triplicate for each
concentration. The secretion of IL-12 and IL-6 was measured by
sandwich ELISA as described by Bhagat L., et al. (Biochem. Biophys.
Res. Commun. 300: 853-861 (2003)). The required reagents, including
cytokine antibodies and standards were purchased from BD
Biosciences Pharmingen (San Diego, Calif.).
Example 5
Mouse Splenomegaly Assay
Female BALB/c mice (4-6 weeks, 19-21 gm) were divided into groups
of three mice. Immunomer compounds were dissolved in sterile
phosphate buffered saline (PBS) and administered subcutaneously
(SC) to mice at a dose of 5 mg/kg. The mice were sacrificed after
48 hr and the spleens were harvested and weighed as described by
Zhao, Q., et al. (Biochem Pharmacol. 51: 173-182 (1996)) and
Branda, R. F., et al. (Biochem. Pharmacol. 45: 2037-2043
(1993)).
Example 6
Activation of the NF-.kappa.B Pathway
Toll-like receptor 9 (TLR9) has been shown to recognize
unmethylated CpG-dinucleotides in bacterial, plasmid and synthetic
DNAs (Hemmi H., et al. Nature 408: 740-745 (2000)) and activate
stress kinase (Yi A. K., et al. J. Immunol. 161: 4493-4497 (1998))
and NF-.kappa.B pathways (Stacey K. J., et al. J. Immunol. 157:
2116-2122 (1996)). NF-.kappa.B activation in J774 cells treated
with immunomer compounds was carried out and analyzed by EMSA as
described Yu D., et al. (Biochem. Biophys. Res. Commun. 297: 83-90
(2002)) and Bhagat L., et al. (Biochem. Biophys. Res. Commun. 300:
853-861 (2003)).
Example 7
Isolation of Human B Cells and Plasmacytoid Dendritic Cells
(pDCs)
PBMCs from freshly drawn healthy volunteer blood (CBR Laboratories,
Boston, Mass.) were isolated by Ficoll density gradient
centrifugation method (Histopaque-1077, Sigma) and B cells were
isolated from PBMCs by positive selection using the CD19 cell
isolation kit (Miltenyi Biotec) according to the manufacturer's
instructions. Table 1 shows the immunostimulatory activity of
immunomer compounds of the invention in C57BL/6 Splenocyte
Assay.
TABLE-US-00001 TABLE I Immunomer Structure and C57BL/6 Splenocyte
Assay (24 hs) SEQ ID NO Sequences and Modification (5'-3') IL-12
IL-12 IL-12 (pg/ml) (pg/ml) (pg/ml) 1 .mu.g/ml 1 .mu.g/ml 1
.mu.g/ml 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 6396
.+-. 10 911 .+-. 61 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 7275 .+-.
77 749 .+-. 80 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 8034 .+-.
14 918 .+-. 136 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 752 .+-. 98 and
37 25 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and
34 26 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
389 .+-. 59 28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 717
.+-. 25 29 5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 849 .+-.
29 30 5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1 TGTCT-5' and 39
Media 75 .+-. 28 104 .+-. 7 IL-6 IL-6 IL-6 (pg/ml) (pg/ml) (pg/ml)
1 .mu.g/ml 1 .mu.g/ml 1 .mu.g/ml 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 2195
.+-. 77 423 .+-. 99 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 3278 .+-. 2
840 .+-. 243 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 7080 .+-. 0
1553 .+-. 670 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 693 .+-. 226 and
37 25 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and
34 26 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
132- 9 .+-. 53 28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 18
.+-. 3 29 5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 1230 .+-.
83 30 5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1 TGTCT-5' and 39
Media 102 .+-. 25 12 .+-. 2 Normal phase represents a
phosphorothioate linkage; o represents a phosphodiester linkage.
G.sub.1 = 2'-deoxy-7-deazaguanosine G.sub.2 = Arabinoguanosine
C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine- X
= Glycerol Linker Y = C3 Linker
Example 8
Human pDC Cultures and IFN-.alpha. and IFN-.beta. ELISA
pDCs were isolated from human PBMCs using a BDCA-4 cell isolation
kit (Miltenyi Biotec) according to the manufacturer's instructions.
pDC were plated in 96-well plates using 1.times.10.sup.6 cells/mL,
200 .mu.L/well). The immunomer compounds were added to a final
concentration of 0.3, 1.0, 3.0, or 10.0 .mu.g/mL to the cell
cultures and incubated at 37.degree. C. for 24 hr. Supernatants
were then harvested and assayed for IFN-.alpha. and IFN-.beta.
using ELISA kit (PBL). Tables 2A-2D show an average .+-.SD of
IFN-.alpha. and IFN-.beta. for immunomer compounds according to the
invention at a concentration of 10.0 .mu.g/mL.
TABLE-US-00002 TABLE 2A Immunomer Structure and Immunostimulatory
Activity in Human DC Assay (24 hs) SEQ ID NO Sequences and
Modification (5'-3') IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN1 DN2 DN3
1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
2611- 2 .+-. 604 25825 .+-. 416 96264 .+-. 605 2
5'-TCG.sub.1AACG.sub.1TTCG-X-GCTTG.sub.1CAAG.sub.1CT-5' 20340 .+-.
106 12270 .+-. 306 105804 .+-. 688 3
5'-TCTCACCTTCT-X-TCTTCCACTCT-5' 185 .+-. 0 311 .+-. 4 1649 .+-. 262
4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
5 5'-TCG.sub.2AACG.sub.2TTCG-X-GCTTG.sub.2CAAG.sub.2CT-5' media 177
.+-. 0 177 .+-. 0 0 .+-. 0 IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN4 DN5 DN6
1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
41718 .+-. 1015 25011 .+-. 5 19608 .+-. 5 2
5'-TCG.sub.1AACG.sub.1TTCG-X-GCTTG.sub.1CAAG.sub.1CT-5' 49176 .+-.
302 14014 .+-. 1414 21988 .+-. 1413 3
5'-TCTCACCTTCT-X-TCTTCCACTCT-5' 0 .+-. 0 197 .+-. 0 201 .+-. 0 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
5 5'-TCG.sub.2AACG.sub.2TTCG-X-GCTTG.sub.2CAAG.sub.2CT-5' media 0
.+-. 0 201 .+-. 0 196 .+-. 0 Normal phase represents a
phosphorothioate linkage G.sub.1 = 2'-deoxy-7-deazaguanosine
G.sub.2 = Arabinoguanosine X = Glycerol linker
TABLE-US-00003 TABLE 2B Immunomer Structure and Immunostimulatory
Activity in Human DC Assay (24 hs) SEQ ID NO Sequences and
Modification (5'-3') IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN1 DN2 DN3
6 5'-TCG.sub.1TCG.sub.1AACG.sub.1TTCG.sub.1AGATGAT-3' 37116 .+-.
1108 44624 .+-. 321 58908 .+-. 707 7
5'-TCG.sub.2TCG.sub.2AACG.sub.2TTCG.sub.2AGATGAT-3' 6606 .+-. 950
5022 .+-. 334 15637 .+-. 698 8
5'-TCG.sub.3TCG.sub.3AACG.sub.3TTCG.sub.3AGATGAT-3' 1405 .+-. 121
7750 .+-. 618 46311 .+-. 506 9
5'-TC.sub.1GTC.sub.1GAAC.sub.1GTTC.sub.1GAGATGAT-3' 611 .+-. 33 231
.+-. 4 0 .+-. 0 10
5'-TC.sub.2GTC.sub.2GAAC.sub.2GTTC.sub.2GAGATGAT-3' 269 .+-. 7 185
.+-. 0 2574 .+-. 64 11
5'-TC.sub.3GTC.sub.3GAAC.sub.3GTTC.sub.3GAGATGAT-3' 191 .+-. 0 188
.+-. 0 0 .+-. 0 media 177 .+-. 0 177 .+-. 0 0 .+-. 0 IFN-.alpha.
(pg/ml) IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10
.mu.g/ml 10 .mu.g/ml DN4 DN5 DN6 6
5'-TCG.sub.1TCG.sub.1AACG.sub.1TTCG.sub.1AGATGAT-3' 98178 .+-. 375
68722 .+-. 1358 31678 .+-. 715 7
5'-TCG.sub.2TCG.sub.2AACG.sub.2TTCG.sub.2AGATGAT-3' 40782 .+-. 885
19180 .+-. 735 8696 .+-. 1122 8
5'-TCG.sub.3TCG.sub.3AACG.sub.3TTCG.sub.3AGATGAT-3' 12446 .+-. 894
42195 .+-. 2665 582 .+-. 78 9
5'-TC.sub.1GTC.sub.1GAAC.sub.1GTTC.sub.1GAGATGAT-3' 10175 .+-. 206
15966 .+-. 1256 6857 .+-. 1335 10
5'-TC.sub.2GTC.sub.2GAAC.sub.2GTTC.sub.2GAGATGAT-3' 13028 .+-. 911
1947 .+-. 204 30 .+-. 5 11
5'-TC.sub.3GTC.sub.3GAAC.sub.3GTTC.sub.3GAGATGAT-3' 0 .+-. 0 355
.+-. 16 17 .+-. 0 media 0 .+-. 0 12 .+-. 0 10 .+-. 0 Normal phase
represents a phosphorothioate linkage G.sub.1 =
2'-deoxy-7-deazaguanosine G.sub.2 = Arabinoguanosine G.sub.3 =
2'-deoxyinosine C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine-
C.sub.2 = Arabinocytidine C.sub.3 = 2'-deoxy-5-hydroxycytidine
TABLE-US-00004 TABLE 2C Immunomer Structure and Immunostimulatory
Activity in Human DC Assay (24 hs) SEQ ID NO Sequences and
Modification (5'-3') IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN1 DN2 DN3
1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
670- 88 .+-. 306 35055 .+-. 659 62805 .+-. 328 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 12588 .+-.
448 19986 .+-. 1418 38002 .+-. 1087 12
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 16090 .+-.
179 16712 .+-. 584 90560 .+-. 1690 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
9092 .+-. 291 9286 .+-. 615 60570 .+-. 867 14
5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 770 .+-. 158 208 .+-. 28 5529 .+-.
286 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
403- 53 .+-. 542 33164 .+-. 419 72730 .+-. 954 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 160 .+-. 7 259 .+-. 20 0 .+-. 0 IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10
.mu.g/ml DN4 DN5 DN6 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
669- 80 .+-. 217 6552 .+-. 1 7992 .+-. 24 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 83115 .+-.
134 12 5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 61230
.+-. 1120 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
344- 30 .+-. 451 14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 2044 .+-. 62 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
337- 16 .+-. 872 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
24- 40 .+-. 23 2403 .+-. 4 16
5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 1316 .+-. 0
1683 .+-. 10 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 546 .+-. 0 0 .+-. 0 0 .+-. 0 IFN-.alpha. (pg/ml) IFN-.alpha.
(pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN7
DN8 DN9 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
31227 .+-. 1341 9777 .+-. 10 10008 .+-. 10 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 12
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
6083 .+-. 184 16
5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 2164 .+-. 4
17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 0 .+-. 0 0 .+-. 0 0 .+-. 0 IFN-.beta. (pg/ml) IFN .beta.
(pg/ml) 10 .mu.g/ml DN8 10 .mu.g/ml DN9 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
137- 0 .+-. 54 650 .+-. 38 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 12
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 0 .+-. 0 0 .+-. 0 Normal phase represents a phosphorothioate
linkage G.sub.1 = 2'-deoxy-7-deazaguanosine G.sub.2 =
Arabinoguanosine C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine- X
= Glycerol Linker
TABLE-US-00005 TABLE 2D Immunomer Structure and Immunostimulatory
Activity in Human DC Assay (24 hs) SEQ ID NO Sequences and
Modification (5'-3') IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN1 DN2 DN3
19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
314- 5 .+-. 4 5808 .+-. 28 22050 .+-. 407 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
471- 0 .+-. 31 5656 .+-. 0 14157 .+-. 10 21
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1 TGTCT-5' and 39
Media 228 .+-. 0 234 .+-. 0 116 .+-. 0 IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10
.mu.g/ml DN4 DN5 DN6 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
161- 00 .+-. 542 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
23768 .+-. 1317 21
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 5824 .+-.
530 2090 .+-. 81 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 9582 .+-.
49 1623 .+-. 108 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 6912 .+-.
157 1577 .+-. 168 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 19570 .+-. 467
2254 .+-. 25 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1 TGTCT-5' and 39
Media 125 .+-. 3 157 .+-. 0 179 .+-. 0 IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10
.mu.g/ml DN7 DN8 DN9 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 1049
.+-. 13 15594 .+-. 48 6024 .+-. 135 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 2230 .+-.
78 6118 .+-. 3 3162 .+-. 189 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 1535 .+-.
130 6680 .+-. 35 1558 .+-. 45 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 16053 .+-. 3815
9502 .+-. 57 6228 .+-. 0 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 6632 .+-. 184 3166
.+-. 242 29 5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 6864 .+-.
394 1146 .+-. 42 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1 TGTCT-5' and 39
Media 157 .+-. 0 82 .+-. 0 94 .+-. 2 IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10
.mu.g/ml DN10 DN11 DN12 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 8154
.+-. 597 31854 .+-. 136 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 6413 .+-.
876 14493 .+-. 613 9642 .+-. 129 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 2688 .+-.
293 4486 .+-. 94 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 7214 .+-. 18
10068 .+-. 31 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' 11474 .+-.
402 and 34 26 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' 375
.+-. 23 and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 5508 .+-. 410
13956 .+-. 355 6009 .+-. 240 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 5599 .+-. 146 11824
.+-. 720 9977 .+-. 1379 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 11946 .+-.
159 31 5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 0
.+-. 0 32 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5'
10032 .+-. 9 and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1 TGTCT-5' 6420 .+-.
139 and 39 Media 80 .+-. 0 101 .+-. 7 0 .+-. 0 Normal phase
represents a phosphorothioate linkage; o represents a
phosphodiester linkage. G.sub.1 = 2'-deoxy-7-deazaguanosine G.sub.2
= Arabinoguanosine C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine- X
= Glycerol Linker Y = C3 Linker
Example 9
Cytokine Analysis
The secretion of IFN-.alpha. in vertebrate cells, preferably BALB/c
mouse spleen cells or human PBMC, was measured by sandwich ELISA.
The required reagents including cytokine antibodies and cytokine
standards were purchased form PharMingen, San Diego, Calif. ELISA
plates (Costar) were incubated with appropriate antibodies at 5
.mu.g/mL in PBSN buffer (PBS/0.05% sodium azide, pH 9.6) overnight
at 4.degree. C. and then blocked with PBS/1% BSA at 37.degree. C.
for 30 minutes. Cell culture supernatants and cytokine standards
were appropriately diluted with PBS/10% FBS, added to the plates in
triplicate, and incubated at 25.degree. C. for 2 hours. Plates were
overlaid with 1 .mu.g/mL appropriate biotinylated antibody and
incubated at 25.degree. C. for 1.5 hours. The plates were then
washed extensively with PBS-T Buffer (PBS/0.05% Tween 20) and
further incubated at 25.degree. C. for 1.5 hours after adding
streptavidin conjugated peroxidase (Sigma, St. Louis, Mo.). The
plates were developed with Sure Blue.TM. (Kirkegaard and Perry)
chromogenic reagent and the reaction was terminated by adding Stop
Solution (Kirkegaard and Perry). The color change was measured on a
Ceres 900 HDI Spectrophotometer (Bio-Tek Instruments).
Human peripheral blood mononuclear cells (PBMCs) were isolated from
peripheral blood of healthy volunteers by Ficoll-Paque density
gradient centrifugation (Histopaque-1077, Sigma, St. Louis, Mo.).
Briefly, heparinized blood was layered onto the Histopaque-1077
(equal volume) in a conical centrifuge and centrifuged at
400.times.g for 30 minutes at room temperature. The buffy coat,
containing the mononuclear cells, was removed carefully and washed
twice with isotonic phosphate buffered saline (PBS) by
centrifugation at 250.times.g for 10 minutes. The resulting cell
pellet was then resuspended in RPMI 1640 medium containing
L-glutamine (MediaTech, Inc., Herndon, Va.) and supplemented with
10% heat inactivated FCS and penicillin-streptomycin (100U/ml).
Cells were cultured in 24 well plates for different time periods at
1.times.10.sup.6 cells/ml/well in the presence or absence of
oligonucleotides. At the end of the incubation period, supernatants
were harvested and stored frozen at -70.degree. C. until assayed
for various cytokines including IFN-.alpha. (BioSource
International) by sandwich ELISA. The results are shown in Table
3A-3D below.
In all instances, the levels of IFN-.alpha. in the cell culture
supernatants was calculated from the standard curve constructed
under the same experimental conditions for IFN-.alpha..
TABLE-US-00006 TABLE 3A Immunomer Structure and Immunostimulatory
Activity in Human PBMC Assay (24 hs) SEQ ID NO Sequences and
Modification (5'-3') IFN-.alpha. IFN-.alpha. IFN-.alpha. (pg/ml)
(pg/ml) (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN4 DN5 DN6 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
8222 .+-. 44 6114 .+-. 1 3604 .+-. 1 2
5'-TCG.sub.1AACG.sub.1TTCG-X-GCTTG.sub.1CAAG.sub.1CT-5' 6700 .+-. 7
6272 .+-. 24 2822 .+-. 24 3 5'-TCTCACCTTCT-X-TCTTCCACTCT-5' 0 .+-.
0 80 .+-. 0 80 .+-. 0 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
5 5'-TCG.sub.2AACG.sub.2TTCG-X-GCTTG.sub.2CAAG.sub.2CT-5' media 0
.+-. 0 78 .+-. 0 83 .+-. 0 IFN-.alpha. IFN-.alpha. IFN-.alpha.
(pg/ml) (pg/ml) (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN1 DN2
DN3 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
7038 .+-. 11 2921 .+-. 321 12514 .+-. 351 2
5'-TCG.sub.1AACG.sub.1TTCG-X-GCTTG.sub.1CAAG.sub.1CT-5' 7332 .+-.
269 3647 .+-. 704 10872 .+-. 613 3 5'-TCTCACCTTCT-X-TCTTCCACTCT-5'
19 .+-. 7 0 .+-. 0 0 .+-. 0 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
5 5'-TCG.sub.2AACG.sub.2TTCG-X-GCTTG.sub.2CAAG.sub.2CT-5' media 33
.+-. 0 0 .+-. 0 0 .+-. 0 Normal phase represents a phosphorothioate
linkage G.sub.1 = 2'-deoxy-7-deazaguanosine G.sub.2 =
Arabinoguanosine X = Glycerol linker
TABLE-US-00007 TABLE 3B Immunomer Structure and Immunostimulatory
Activity in Human PBMC Assay (24 hs) SEQ ID NO Sequences and
Modification (5'-3') IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN1 DN2 DN3
6 5'-TCG.sub.1TCG.sub.1AACG.sub.1TTCG.sub.1AGATGAT-3' 3487 .+-.
1015 268 .+-. 3 3883 .+-. 50 7
5'-TCG.sub.2TCG.sub.2AACG.sub.2TTCG.sub.2AGATGAT-3' 9 .+-. 0 30
.+-. 0 0 .+-. 0 8
5'-TCG.sub.3TCG.sub.3AACG.sub.3TTCG.sub.3AGATGAT-3' 126 .+-. 1 0
.+-. 0 0 .+-. 0 9
5'-TC.sub.1GTC.sub.1GAAC.sub.1GTTC.sub.1GAGATGAT-3' 0 .+-. 0 0 .+-.
0 0 .+-. 0 10 5'-TC.sub.2GTC.sub.2GAAC.sub.2GTTC.sub.2GAGATGAT-3' 0
.+-. 0 20 .+-. 0 0 .+-. 0 11
5'-TC.sub.3GTC.sub.3GAAC.sub.3GTTC.sub.3GAGATGAT-3' 11 .+-. 1 5
.+-. 0 76 .+-. 0 media 33 .+-. 0 0 .+-. 0 0 .+-. 0 IFN-.alpha.
(pg/ml) IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10
.mu.g/ml 10 .mu.g/ml DN4 DN5 DN6 6
5'-TCG.sub.1TCG.sub.1AACG.sub.1TTCG.sub.1AGATGAT-3' 1950 .+-. 88
4342 .+-. 225 426 .+-. 85 7
5'-TCG.sub.2TCG.sub.2AACG.sub.2TTCG.sub.2AGATGAT-3' 0 .+-. 0 1807
.+-. 0 31 .+-. 15 8
5'-TCG.sub.3TCG.sub.3AACG.sub.3TTCG.sub.3AGATGAT-3' 0 .+-. 0 2876
.+-. 344 48 .+-. 5 9
5'-TC.sub.1GTC.sub.1GAAC.sub.1GTTC.sub.1GAGATGAT-3' 0 .+-. 0 5 .+-.
0 4 .+-. 0 10 5'-TC.sub.2GTC.sub.2GAAC.sub.2GTTC.sub.2GAGATGAT-3' 0
.+-. 0 8 .+-. 0 5 .+-. 3 11
5'-TC.sub.3GTC.sub.3GAAC.sub.3GTTC.sub.3GAGATGAT-3' 0 .+-. 0 2111
.+-. 330 11 .+-. 3 media 0 .+-. 0 48 .+-. 9 11 .+-. 2 Normal phase
represents a phosphorothioate linkage G.sub.1 =
2'-deoxy-7-deazaguanosine G.sub.2 = Arabinoguanosine G.sub.3 =
2'-deoxyinosine C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine-
C.sub.2 = Arabinocytidine C.sub.3 = 2'-deoxy-5-hydroxycytidine
TABLE-US-00008 TABLE 3C Immunomer Structure and Immunostimulatory
Activity in Human PBMC Assay (24 hs) SEQ ID NO Sequences and
Modification (5'-3') IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN1 DN2 DN3
1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
208 .+-. 33 432 .+-. 5 1345 .+-. 20 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 11 .+-. 1
59 .+-. 0 173 .+-. 41 12
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 16 .+-. 1 55
.+-. 7 324 .+-. 49 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
882 .+-. 32 733 .+-. 80 2035 .+-. 16 14
5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 50 .+-. 27 39 .+-. 17 4 .+-. 0 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
604 .+-. 6 465 .+-. 70 1902 .+-. 30 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 20 .+-. 4 12 .+-. 0 3 .+-. 0 IFN-.alpha. (pg/ml) IFN-.alpha.
(pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN4
DN5 DN6 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
900 .+-. 8 432 .+-. 26 582 .+-. 20 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 92 .+-. 6
12 5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 441 .+-.
76 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
732 .+-. 8 14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 19 .+-. 5 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
880 .+-. 8 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
27 .+-. 0 26 .+-. 0 16
5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 19 .+-. 0
23 .+-. 0 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 6 .+-. 1 0 .+-. 0 0 .+-. 0 IFN-.alpha. (pg/ml) IFN-.alpha.
(pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN7
DN8 DN9 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
324 .+-. 18 578 .+-. 28 741 .+-. 25 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 12
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
6 .+-. 0 16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5'
0 .+-. 0 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 0 .+-. 0 0 .+-. 0 0 .+-. 0 Normal phase represents a
phosphorothioate linkage G.sub.1 = 2'-deoxy-7-deazaguanosine
G.sub.2 = Arabinoguanosine C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine- X
= Glycerol Linker
TABLE-US-00009 TABLE 3D Immunomer Structure and Immunostimulatory
Activity in Human PBMC Assay (24 hs) SEQ ID NO Sequences and
Modifications (5'-3') IFN-.alpha. (pg/ml) IFN-.alpha. (pg/ml)
IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN1 DN2 DN3
19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
8 .+-. 0 65 .+-. 3 314 .+-. 23 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
9 .+-. 0 10 .+-. 2 487 .+-. 87 21
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1 TGTCT-5' and 39
Media 11 .+-. 0 10 .+-. 0 0 .+-. 0 IFN-.alpha. (pg/ml) IL-10
(pg/ml) IL-10 (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN4 DN5
DN6 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
144- 6 .+-. 7 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
942 .+-. 1 21
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 126 .+-. 2
159 .+-. 13 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 239 .+-. 23
356 .+-. 109 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 147 .+-. 23
185 .+-. 46 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 107 .+-. 15 148
.+-. 37 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1 TGTCT-5' and 39
Media 0 .+-. 0 68 .+-. 5 67 .+-. 0 IL-10 (pg/ml) IFN-.alpha.
(pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml DN7
DN8 DN9 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 242 .+-.
1 549 .+-. 37 9 .+-. 0 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 241 .+-. 2
250 .+-. 12 14 .+-. 1 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 238 .+-. 0
224 .+-. 25 8 .+-. 1 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 238 .+-. 0 668
.+-. 10 41 .+-. 7 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 233 .+-. 31 12
.+-. 0 29 5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 47 .+-. 4 5
.+-. 1 30 5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5'
31 5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1 TGTCT-5' and 39
Media 0 .+-. 0 0 .+-. 0 2 .+-. 0 IFN-.alpha. (pg/ml) IFN-.alpha.
(pg/ml) IFN-.alpha. (pg/ml) 10 .mu.g/ml 10 .mu.g/ml 10 .mu.g/ml
DN10 DN11 DN12 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 232 .+-.
8 252 .+-. 16 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 67 .+-. 1
195 .+-. 3 364 .+-. 8 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 70 .+-. 1
148 .+-. 3 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 443 .+-. 29 678
.+-. 133 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' 298 .+-. 16
and 34 26 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' 12 .+-.
1 and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 2 .+-. 0 94 .+-. 5
512 .+-. 33 29 5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 3 .+-.
0 61 .+-. 18 168 .+-. 25 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 992 .+-. 2
31 5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 9 .+-. 0
32 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' 1528
.+-. 20 and 38 33 5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1
TGTCT-5' 69 .+-. 10 and 39 Media 0 .+-. 0 6 .+-. 0 7 .+-. 0 Normal
phase represents a phosphorothioate linkage; o represents a
phosphodiester linkage. G.sub.1 = 2'-deoxy-7-deazaguanosine G.sub.2
= Arabinoguanosine C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine- X
= Glycerol Linker Y = C3 Linker
Example 10
Flow Cytometric Analysis
Cell surface markers of CD69 and CD86 were detected with a Coulter
Epics-XL Flow Cytometer using anti-human CD69-Fitc and CD86-Fitc,
which were purchased from BD Pharmingen (San Diego, USA). Staining
methods were briefly descried as follow. The activated culture
cells were blocked with 10% Human AB serum (Sigma) in staining
buffer (PBS with 1% BSA and 0.1% NaN.sub.3) at 4.degree. C. for 1
hour and stained with the antibodies at 4.degree. C. overnight.
PBMCs (4.times.10.sup.5) were stained with CD69-Fitc and CD86-Fitc.
PDCs (2.times.10.sup.5) were stained CD86-Fitc. The cell staining
data were acquired and analyzed with Coulter System II software
(see Tables 4A-4F below).
TABLE-US-00010 TABLE 4A Immunomer Structure and Expression of BC
from Human PBMC (2 .times. 10.sup.6 cell/ml) (24 hs) SEQ ID NO
Sequences and Modification (5'-3') % CD86 % CD86 % CD86 1 .mu.g/ml
1 .mu.g/ml 1 .mu.g/ml DN1 DN2 DN3 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
36.4- 17.7 36.4 2
5'-TCG.sub.1AACG.sub.1TTCG-X-GCTTG.sub.1CAAG.sub.1CT-5' 27.2 6.3
30.4 3 5'-TCTCACCTTCT-X-TCTTCCACTCT-5' 15 7.3 11.3 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
5 5'-TCG.sub.2AACG.sub.2TTCG-X-GCTTG.sub.2CAAG.sub.2CT-5' media
10.7 8 7.6 % CD86 % CD86 % CD86 1 .mu.g/ml 1 .mu.g/ml 1 .mu.g/ml
DN4 DN5 DN6 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
33.3- 12.9 27.6 2
5'-TCG.sub.1AACG.sub.1TTCG-X-GCTTG.sub.1CAAG.sub.1CT-5' 25 17.5
32.4 3 5'-TCTCACCTTCT-X-TCTTCCACTCT-5' 10 21.3 17.6 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
5 5'-TCG.sub.2AACG.sub.2TTCG-X-GCTTG.sub.2CAAG.sub.2CT-5' media 5.2
5.4 11.8 % CD69 % CD69 % CD69 1 .mu.g/ml 1 .mu.g/ml 1 .mu.g/ml DN1
DN2 DN3 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
27.5- 61 75.8 2
5'-TCG.sub.1AACG.sub.1TTCG-X-GCTTG.sub.1CAAG.sub.1CT-5' 52.5 46.5
69.7 3 5'-TCTCACCTTCT-X-TCTTCCACTCT-5' 0 11.8 8.5 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
5 5'-TCG.sub.2AACG.sub.2TTCG-X-GCTTG.sub.2CAAG.sub.2CT-5' media 0
11.1 11.1 % CD69 % CD69 % CD69 1 .mu.g/ml 1 .mu.g/ml 1 .mu.g/ml DN4
DN5 DN6 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
58.3- 51.8 39.1 2
5'-TCG.sub.1AACG.sub.1TTCG-X-GCTTG.sub.1CAAG.sub.1CT-5' 62.5 56.5
43.6 3 5'-TCTCACCTTCT-X-TCTTCCACTCT-5' 0 31.1 16.9 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
5 5'-TCG.sub.2AACG.sub.2TTCG-X-GCTTG.sub.2CAAG.sub.2CT-5' media 5.2
18.9 8.9 Normal phase represents a phosphorothioate linkage G.sub.1
= 2'-deoxy-7-deazaguanosine G.sub.2 = Arabinoguanosine X = Glycerol
linker
TABLE-US-00011 TABLE 4B Immunomer Structure and Expression of BC
from Human PBMC (2 .times. 10.sup.6 cell/ml) (24 hs) SEC ID NO
Sequences and Modification (5'-3') % CD86 % CD86 % CD86 1 .mu.g/ml
DN1 1 .mu.g/ml DN2 1 .mu.g/ml DN3 6
5'-TCG.sub.1TCG.sub.1AACG.sub.1TTCG.sub.1AGATGAT-3' 43.4 25 34.6 7
5'-TCG.sub.2TCG.sub.2AACG.sub.2TTCG.sub.2AGATGAT-3' 46.7 42.6 47.6
8 5'-TCG.sub.3TCG.sub.3AACG.sub.3TTCG.sub.3AGATGAT-3' 41.1 25.7
38.5 9 5'-TC.sub.1GTC.sub.1GAAC.sub.1GTTC.sub.1GAGATGAT-3' 25 20.8
27.6 10 5'-TC.sub.2GTC.sub.2GAAC.sub.2GTTC.sub.2GAGATGAT-3' 36.4
22.2 26 11 5'-TC.sub.3GTC.sub.3GAAC.sub.3GTTC.sub.3GAGATGAT-3' 30
17 22.2 media 10.7 8 7.6 % CD86 % CD86 % CD86 1 .mu.g/ml DN4 1
.mu.g/ml DN5 1 .mu.g/ml DN6 6
5'-TCG.sub.1TCG.sub.1AACG.sub.1TTCG.sub.1AGATGAT-3' 40 43.5 24.8 7
5'-TCG.sub.2TCG.sub.2AACG.sub.2TTCG.sub.2AGATGAT-3' 36.4 41 36.2 8
5'-TCG.sub.3TCG.sub.3AACG.sub.3TTCG.sub.3AGATGAT-3' 57.1 30.4 34.9
9 5'-TC.sub.1GTC.sub.1GAAC.sub.1GTTC.sub.1GAGATGAT-3' 13.3 12.1
15.2 10 5'-TC.sub.2GTC.sub.2GAAC.sub.2GTTC.sub.2GAGATGAT-3' 25 14.4
16.3 11 5'-TC.sub.3GTC.sub.3GAAC.sub.3GTTC.sub.3GAGATGAT-3' 18.1 15
16.8 media 5.2 3.9 6.8 % CD69 % CD69 % CD69 1 .mu.g/ml DN1 1
.mu.g/ml DN2 1 .mu.g/ml DN3 6
5'-TCG.sub.1TCG.sub.1AACG.sub.1TTCG.sub.1AGATGAT-3' 56.4 43.8 68.7
7 5'-TCG.sub.2TCG.sub.2AACG.sub.2TTCG.sub.2AGATGAT-3' 55.6 58.6
65.5 8 5'-TCG.sub.3TCG.sub.3AACG.sub.3TTCG.sub.3AGATGAT-3' 50 39.3
73.1 9 5'-TC.sub.1GTC.sub.1GAAC.sub.1GTTC.sub.1GAGATGAT-3' 15.4 27
20 10 5'-TC.sub.2GTC.sub.2GAAC.sub.2GTTC.sub.2GAGATGAT-3' 20 31.4
31.5 11 5'-TC.sub.3GTC.sub.3GAAC.sub.3GTTC.sub.3GAGATGAT-3' 10 22.2
24.3 media 0 11.1 11.1 % CD69 % CD69 % CD69 1 .mu.g/ml DN4 1
.mu.g/ml DN5 1 .mu.g/ml DN6 6
5'-TCG.sub.1TCG.sub.1AACG.sub.1TTCG.sub.1AGATGAT-3' 57.1 7
5'-TCG.sub.2TCG.sub.2AACG.sub.2TTCG.sub.2AGATGAT-3' 60 8
5'-TCG.sub.3TCG.sub.3AACG.sub.3TTCG.sub.3AGATGAT-3' 37.5 9
5'-TC.sub.1GTC.sub.1GAAC.sub.1GTTC.sub.1GAGATGAT-3' 15.4 10
5'-TC.sub.2GTC.sub.2GAAC.sub.2GTTC.sub.2GAGATGAT-3' 11.1 11
5'-TC.sub.3GTC.sub.3GAAC.sub.3GTTC.sub.3GAGATGAT-3' 14.3 media 5.2
Normal phase represents a phosphorothioate linkage G.sub.1 =
2'-deoxy-7-deazaguanosine G.sub.2 = Arabinoguanosine G.sub.3 =
2'-deoxyinosine C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine-
C.sub.2 = Arabinocytidine C.sub.3 = 2'-deoxy-5-hydroxycytidine
TABLE-US-00012 TABLE 4C Immunomer Structure and Expression of BC
from Human PBMC (2 .times. 10.sup.6 cell/ml) (24 hs) SEQ ID NO
Sequences and Modification (5'-3') % CD86 % CD86 % CD86 1 .mu.g/ml
DN1 1 .mu.g/ml DN2 1 .mu.g/ml DN3 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
32.- 3 34.8 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 41.4 51.6
12 5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 33.3 51.5
13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
20 - 25.6 14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 31.1 26.1 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
17.- 1 23.9 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 19.4 20.9 % CD86 % CD86 % CD86 1 .mu.g/ml DN4 1 .mu.g/ml DN5
1 .mu.g/ml DN6 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
15- .4 33.3 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 12
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
30- .7 45.4 16
5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 30 41.6 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 8.6 2.7 % CD86 % CD86 % CD86 1 .mu.g/ml DN7 1 .mu.g/ml DN8 1
.mu.g/ml DN9 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
35.- 5 23.5 17.6 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 12
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
56.- 5 16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5'
46.7 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 9 20 15.3 % CD69 % CD69 % CD69 1 .mu.g/ml DN1 1 .mu.g/ml DN2
1 .mu.g/ml DN3 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
23.- 5 64 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 20.8 62.5
12 5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 13.6 59 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
12.- 5 46.4 14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15.9 52.9 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
12.- 2 51.6 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 14.8 34 % CD69 % CD69 % CD69 1 .mu.g/ml DN4 1 .mu.g/ml DN5 1
.mu.g/ml DN6 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
53- .8 62.5 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 12
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
77- .7 70.6 16
5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 57.1 64.7
17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 26.3 15 % CD69 % CD69 % CD69 1 .mu.g/ml DN7 1 .mu.g/ml DN8 1
.mu.g/ml DN9 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
28.- 6 50 25 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 12
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
70.- 6 16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5'
72.7 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 14.1 13.2 12 Normal phase represents a phosphorothioate
linkage G.sub.1 = 2'-deoxy-7-deazaguanosine G.sub.2 =
Arabinoguanosine C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine- X
= Glycerol Linker
TABLE-US-00013 TABLE 4D Immunomer Structure and Expression of BC
from Human PBMC (2 .times. 10.sup.6 cell/ml) (24 hs) SEQ ID NO
Sequences and Modification (5'-3') % CD86 % CD86 % CD86 1 .mu.g/ml
DN1 1 .mu.g/ml DN2 1 .mu.g/ml/DN3 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
64- .3 57.2 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 59.2 58.3
12 5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 49.3 40.9
13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
25- .3 24.7 14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15.4 17.2 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
30- .6 23.7 4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 2.6 13.9 % CD86 % CD86 % CD86 1 .mu.g/ml DN4 1 .mu.g/ml DN5 1
.mu.g/ml DN6 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
35.- 9 30.3 35.6 40
5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5' 57.9 12
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 34.9 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 12.3 11.1 14 % CD86 % CD86 % CD86 1 .mu.g/ml DN7 1 .mu.g/ml
DN8 1 .mu.g/ml DN9 1
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
28- 32.3 40 5'-TCG.sub.1AACG.sub.1TTCT-X-TCTTG.sub.1CAAG.sub.1CT-5'
12 5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 13
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
14 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 15
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
4
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
16 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 17
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
18
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
media 10.9 12.6 Normal phase represents a phosphorothioate linkage
G.sub.1 = 2'-deoxy-7-deazaguanosine G.sub.2 = Arabinoguanosine
C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine- X
= Glycerol Linker
TABLE-US-00014 TABLE 4E Immunomer Structure and Expression of BC
from Human PBMC assay (24 hs) SEQ ID NO Sequences and Modification
(5'-3') % CD86 % CD86 % CD86 1 .mu.g/ml DN1 1 .mu.g/ml DN2 1
.mu.g/ml DN3 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
20 - 9 34.6 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21.- 7 12.5 31.4 21
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' and 39
Media 8.7 % CD86 % CD86 % CD86 1 .mu.g/ml DN4 1 .mu.g/ml DN5 1
.mu.g/ml DN6 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
42.- 3 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
72.- 7 21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5'
14.5 17.1 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 27.8 28.6
and 35 23 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5'
28.9 22.2 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 23.2 21.8 and 37
25 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34
26 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' and 39
Media 5.9 4.0 6.0 % CD86 % CD86 % CD86 1 .mu.g/ml DN7 1 .mu.g/ml
DN8 1 .mu.g/ml DN9 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
- 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 65 46.3
40.3 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 75 49.2
46.5 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 78.9 54.3
45 and 36 24 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 83.3
33.8 29.4 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 22.5 35.6 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 43.1 47.8 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' Media 4.6
0 10.5 % CD86 % CD86 % CD86 1 .mu.g/ml 1 .mu.g/ml 1 .mu.g/ml DN10
DN11 DN12 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
- 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 24.4
60.7 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 23.9 61.5
53.8 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 32.8 72 and
36 24 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 22.7 52.6
and 37 25 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5'
31.7 and 34 26 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5'
23.9 and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 21 57.1 29.6 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 34.7 63.1 43.5 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 24.5 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 28.6 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' 44.2 and 38
33 5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' 28.3
and 39 Media 19 8.6 18 % CD69 % CD69 % CD69 1 .mu.g/ml DN1 1
.mu.g/ml DN2 1 .mu.g/ml DN3 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
13 - 22.2 19.2 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
52 - 30.7 59.3 21
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' and 38 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 39 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' Media 3 %
CD69 % CD69 % CD69 1 .mu.g/ml DN4 1 .mu.g/ml DN5 1 .mu.g/ml DN6 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
76 - 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
85.- 1 21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5'
35 20 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 57.3 39.4
and 35 23 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5'
60.4 54.2 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 69 30.2 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' and 39
Media 10 5.9 10.1 % CD69 % CD69 % CD69 1 .mu.g/ml DN7 1 .mu.g/ml
DN8 1 .mu.g/ml DN9 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
- 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 88.2
47.7 59.7 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 97 55 63.3
and 35 23 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5'
96.8 68.3 60.2 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 91.9 40.3 41.9
and 37 25 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5'
and 34 26 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37
27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 36 51.2 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 51.6 66.7 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' and 39
Media 9 10.3 11.1 % CD69 % CD69 % CD69 1 .mu.g/ml 1 .mu.g/ml 1
.mu.g/ml DN10 DN11 DN12 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
- 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 24 61.7
and 34 22 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5'
29.2 71.4 58 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 35.7 60.5
and 36 24 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 32.2
62.9 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' 23.6 and 34
26 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' 16.7 and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 22.1 50 42.5 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 31.5 70.5 54.4 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 19.5 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 15.5 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' 40 and 38
33 5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' 19.3
and 39 Media 13.4 13.4 12.9 Normal phase represents a
phosphorothioate linkage; o represents a phosphodiester linkage.
G.sub.1 = 2'-deoxy-7-deazaguanosine G.sub.2 = Arabinoguanosine
C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine- X
= Glycerol Linker Y = C3 Linker
TABLE-US-00015 TABLE 4F Immunomer Structure and Expression of DC
from Human PBMC assay (24 hs) SEQ ID NO Sequences and Modification
(5'-3') % CD86 % CD86 % CD86 1 .mu.g/ml DN1 1 .mu.g/ml DN2 1
.mu.g/ml DN3 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
1- 1.9 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
1- 2.5 21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5'
and 34 22 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5'
and 35 23 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5'
and 36 24 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' and 37
25 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34
26 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' and 39
Media 13.7 % CD86 % CD86 % CD86 1 .mu.g/ml DN4 1 .mu.g/ml DN5 1
.mu.g/ml DN6 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
- 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 54.7 68
and 34 22 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5'
58.8 75.3 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 60.3 73.4
and 36 24 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 51.8
61.1 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' and 39
Media 33.7 62.8 % CD86 % CD86 % CD86 1 .mu.g/ml DN7 1 .mu.g/ml DN8
1 .mu.g/ml DN9 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
- 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 45.4
88.7 78.3 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 54.9 89.3
79.1 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 55.3 88.6
79.9 and 36 24 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 47
85.7 n/a and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 82.1 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 89 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' and 39
Media 47.5 56.1 53.2 % CD86 % CD86 % CD86 1 .mu.g/ml 1 .mu.g/ml 1
.mu.g/ml DN10 DN11 DN12 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
- 20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 72 86.3
and 34 22 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5'
74.4 88.1 81.8 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 77.1 87.5
and 36 24 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 68.3
83.8 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' 60.4 and 34
26 5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' 37.4 and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 61.1 79.6 58.2 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 68.2 87.3 69.5 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 60.3 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 44.7 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' 65.8 and 38
33 5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' 49.1
and 39 Media 69.6 58.3 35.8 Normal phase represents a
phosphorothioate linkage; o represents a phosphodiester linkage.
G.sub.1 = 2'-deoxy-7-deazaguanosine G.sub.2 = Arabinoguanosine
C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine- X
= Glycerol Linker; Y = C3 Linker
Example 11
B cell proliferation assay
A total of 1.times.10.sup.5 B cells/200 .mu.l were stimulated with
0.3, 1.0, 3.0, or 10.0 .mu.g/mL concentrations of immunomer
compounds of the invention for 16 hr, then pulsed with 0.75 .mu.Ci
of [.sup.3H]-thymidine and harvested 8 h later. The incorporation
of radioactivity was measured using liquid scintillation counter.
Table 5 shows an average .+-.SD of B cell proliferation at a final
concentration of 1.0 .mu.g/mL.
TABLE-US-00016 TABLE 5 Immunomer Structure and Immunostimulatory
Activity in Human B-Cell Proliferation Assay (24 hs) SEQ ID NO
Sequences and Modification (5'-3') [.sup.3H]T [.sup.3H]T [.sup.3H]T
(cpm) (cpm) (cpm) 1 .mu.g/ml 1 .mu.g/ml 1 .mu.g/ml DN4 DN5 DN6 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 31127
.+-. 6800 17626 .+-. 2809 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 33368 .+-.
1364 17131 .+-. 1366 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 30845 .+-.
2514 13826 .+-. 2331 and 36 24
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5' 34077 .+-. 3636
8073 .+-. 583 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' and 39
Media 646 .+-. 236 457 .+-. 121 [.sup.3H]T [.sup.3H]T [.sup.3H]T
(cpm) (cpm) (cpm) 1 .mu.g/ml 1 .mu.g/ml 1 .mu.g/ml DN7 DN8 DN9 19
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
20
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
21 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.1CTGTCT-5' 37731
.+-. 2901 and 34 22
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTG.sub.2CTGTCT-5' 38405 .+-.
8056 and 35 23
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTGC.sub.1TGTCT-5' 34702 .+-.
6196 and 36 24 5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-TCTTCCACTCT-5'
23030 .+-. 1941 and 37 25
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTG.sub.1CTGTCT-5' and 34 26
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-Y-TCTTCCACTCT-5' and 37 27
5'-TG.sub.1CAAG.sub.1CTTG.sub.1C-X-CG.sub.1TTCG.sub.1AACG.sub.1T-5'
28 5'-CTGTCG.sub.2TTCTCo-X-oCTCTTG.sub.2CTGTC-5' 29
5'-CTGTCoG.sub.2TTCTC-X-CTCTTG.sub.2oCTGTC-5' 30
5'-TCG.sub.1TGTCG.sub.1TTT-X-TTTG.sub.1CTGTG.sub.1CT-5' 31
5'-TG.sub.1CTGTG.sub.1CTTT-X-TTTCG.sub.1TGTCG.sub.1T-5' 32
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-Y-GACAG.sub.1CTGTCT-5' and 38 33
5'-TG.sub.1CAACG.sub.1CTTG.sub.1C-Y-GACACG.sub.1TGTCT-5' and 39
Media 658 .+-. 205 Normal phase represents a phosphorothioate
linkage; o represents a phosphodiester linkage. G.sub.1 =
2'-deoxy-7-deazaguanosine G.sub.2 = Arabinoguanosine C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurine- X
= Glycerol Linker Y = C3 Linker
EQUIVALENTS
While the foregoing invention has been described in some detail for
purposes of clarity and understanding, it will be appreciated by
one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention and appended claims.
SEQUENCE LISTINGS
1
41 1 11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 1 tcnaacnttc n 11 2 11 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 2 tcnaacnttc g 11 3 11 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 3
tctcaccttc t 11 4 11 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 4 tcnaacnttc n 11 5
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 5 tcnaacnttc g 11 6 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 6 tcntcnaacn ttcnagatga t 21 7 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 7 tcntcnaacn ttcnagatga t 21 8 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 8 tcntcnaacn ttcnagatga t 21 9 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 9 tngtngaang ttngagatga t 21 10 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 10 tngtngaang ttngagatga t 21 11 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 11 tngtngaang ttngagatga t 21 12 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 12 tcnaacnttc 10 13 11 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 13
tcnttcnaac n 11 14 11 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 14 tccaaccttc g 11 15
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 15 tcnttncaac n 11 16 11 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 16 tcnaacnttc t 11 17 11 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 17
tcnaacnttc n 11 18 11 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 18 tcnaangttc n 11 19
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 19 tcnaacnttc n 11 20 11 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 20 tcnaangttc n 11 21 11 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 21
tcnaacnttc n 11 22 11 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 22 tcnaacnttc n 11 23
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 23 tcnaacnttc n 11 24 11 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 24 tcnaacnttc n 11 25 11 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 25
tncaancttn c 11 26 11 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 26 tncaancttn c 11 27
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 27 tncaancttn c 11 28 11 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 28 ctgtcnttct c 11 29 11 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 29
ctgtcnttct c 11 30 11 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 30 tcntgtcntt t 11 31
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 31 tnctgtnctt t 11 32 11 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 32 tcnaacnttc n 11 33 12 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 33
tncaacnctt nc 12 34 11 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 34 tctgtcnttc t 11 35
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 35 tctgtcnttc t 11 36 11 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 36 tctgtngttc t 11 37 11 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 37
tctcaccttc t 11 38 11 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 38 tctgtcnaca g 11 39
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 39 tctgtncaca g 11 40 11 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 40 tcnaacnttc t 11 41 11 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 41
tcgaacgttc g 11
* * * * *